Department of Physiology and Pharmacology, Center for the
Neurobiological Investigation of Drug Abuse, Wake Forest University
School of Medicine, Winston-Salem, North Carolina (S.R.L., H.R.S.,
L.J.P., B.A.B., T.S., S.R.C.); and Department of Chemistry, State
University of New York at Buffalo, Buffalo, New York (H.M.L.D.)
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Introduction |
A
number of studies suggest that the reinforcing and psychostimulant
properties of cocaine are predominantly mediated by its ability to bind
to the dopamine transporter and inhibit dopamine uptake in brain (Ritz
et al., 1987
; Bergman et al., 1989; Giros et al., 1996). A necessary
step in the characterization of the dopamine transporter is the use of
high-affinity selective radioligands. Although
[3H]cocaine itself has been used for this
purpose (Reith et al., 1980; Kennedy and Hanbauer, 1983), its affinity
for dopamine transporters is too low to allow for extensive
characterization. Several nontropane radioligands have been used
successfully to label dopamine transporters, including
[3H]mazindol (Javitch et al., 1984),
[3H]GBR 12783 (Bonnet et al., 1988) and
[3H]GBR 12935 (Richfield, 1991).
However, the binding properties of such ligands at dopamine
transporters may differ from those of cocaine-like tropane structures
(Madras et al., 1989a).
Higher affinity tropane radioligands structurally related to cocaine
have included [3H]WIN 35,428 (Madras et al.,
1989a),
[125I]3
-[4-iodophenyl]-tropane-2-carboxylic
acid methyl ester ([125I]RTI-55; Boja et al.,
1991), and [125I]RTI-121 (Boja et al.,
1992). These ligands have been used autoradiographically to provide
neuroanatomical localization of biogenic amine transport sites in brain
(Canfield et al., 1990; Boja et al., 1992), and to assess changes in
dopamine transporter density in disease states, such as drug abuse
(Little et al., 1993; Staley et al., 1994; Wilson et al., 1994) and
Parkinson's disease (Fischman et al., 1998). These tropane
radioligands have generally exhibited two-site binding properties at
the dopamine transporter (Madras et al., 1989a; Boja et al., 1991,
1992).
To define the cocaine pharmacophore, a number of novel tropane analogs
have been prepared using cocaine as starting material (Boja et al.,
1990; Kosikowski et al., 1992; Carroll et al., 1993; Kelkar et al.,
1994). To increase synthetic flexibility, however, a novel method of
synthesis was developed using vinylcarbenoid precursors as starting
materials (Davies et al., 1991). This synthetic route created a number
of unique tropane analogs, with varying degrees of specificity and
potencies at dopamine, serotonin, and norepinephrine transport sites
(Davies et al. 1993, 1994; Bennett et al., 1995). These compounds
contain different substituents at the 3-position of the tropane ring,
but all share the common structural characteristic of a substituted
(either ethyl- or methyl-) ketone in the 2-position. One of these
compounds is PTT [2-propanoyl-3-(4-tolyl) tropane], a
relatively potent analog with selectivity for dopamine transporters. A
number of studies have characterized the in vivo properties of PTT and
have shown that it increases dopamine levels in nucleus accumbens
(Hemby et al., 1995), whereas it increases locomotor activity for
prolonged periods of time compared with cocaine (Porrino et al., 1994,
1995). Moreover, PTT appears to have different effects in monkeys and
rodents, with high reinforcement efficacy in a progressive ratio test
in rats (Roberts et al., 1999) and low reinforcement efficacy in rhesus
monkeys (Nader et al., 1997), where it blocks cocaine
self-administration for several hours after a single injection.
To more fully characterize the actions of PTT, we have recently
prepared the 3H form of its active enantiomer for
use in dopamine transporter binding assays. This represents the first
use of a radiolabeled 2-ketone-substituted tropane and allows for a
better characterization of the in vitro properties of PTT to compare
with its well established in vivo effects. In this study, we report the
binding of [3H]PTT to dopamine transporters in
membranes and the autoradiographic distribution of
[3H]PTT-binding sites in rat brain.
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Experimental Procedures |
Materials.
[125I]RTI-55 (2200 Ci/mmol) was obtained from New England Nuclear Corp. (Boston, MA).
[3H]PTT (85 Ci/mmol) was synthesized from its
N-demethylated precursor by American Radiolabeled Chemicals
(St. Louis, MO). Sprague-Dawley rats used for homogenate studies were
purchased from Zivic-Miller (Zeleinople, PA) and from Harlan
(Indianapolis, IN) for autoradiographic studies. (
)-Cocaine was
provided by the Research Technology Branch of the National Institute on
Drug Abuse. Buffers and other chemicals for binding studies were
reagent grade chemicals from Sigma and Fisher. Chemicals for the
organic syntheses were obtained from Aldrich (Milwaukee, WI). Hyperfilm
3H was obtained from Amersham (Arlington Heights,
IL) and ART-123 tritium standards from American Radiolabeled Chemicals.
Autoradiograms were analyzed with the computerized image processing
system MCID (Imaging Research, St. Catherine's, Ontario, Canada).
Chemistry.
The N-demethylated precursors for PTT
and other tropane analogs were prepared by copper-catalyzed
1,4-addition of Grignard reagents to the appropriate 
,
-unsaturated ketones. The , -unsaturated ketones were prepared
by the general four-step sequence as described previously (Davies et
al., 1991): rhodium(II) pivalate-catalyzed decomposition of the
appropriate vinyl diazomethane in the presence of
N-(tert-butoxycarbonyl)pyrrole, catalytic
hydrogenation with Wilkinson's catalyst, trifluoroacetic acid-induced
hydrolysis of the tert-butoxycarbonyl protecting group, and
reductive methylation with formaldehyde and sodium cyanoborohydride.
[3H]PTT was prepared from the active enantiomer
of its N-demethylated precursor using
[3H]methyl iodide.
Radioligand Binding in Membranes.
Binding of
[125I]RTI-55 to rat striatal membranes was
conducted by the method of Boja et al. (1991). Male Sprague-Dawley rats (200-250 g) were decapitated by guillotine, and striata were dissected on ice. Tissue was homogenized in 10 volumes of assay buffer (0.32 M
sucrose, 10 mM sodium phosphate buffer, pH 7.4) with a Polytron (setting 6, 20 s), and centrifuged three times at
48,000g for 10 min, with fresh buffer resuspension for each
centrifugation. For [125I]RTI-55, binding
assays were performed in tubes containing 0.5 mg (original wet weight)
of membranes, 0.02 nM [125I]RTI-55, and various
concentrations of unlabeled drugs in a final volume of 2 ml. Tubes were
incubated for 50 min at 25°C. For [3H]PTT,
binding assays contained 4 mg (original wet weight) of membranes, 0.3 nM [3H]PTT, in a final volume of 2 ml. Tubes
were incubated for 30 min at 25°C. For both radioligands, the
reaction was terminated by rapid filtration with 3 × 5 ml of cold
Tris buffer through Whatman GF/B glass fiber filters. Radioactivity was
determined by liquid scintillation spectrophotometry after overnight
extraction of filters in Scinti-Safe scintillation fluid (Fisher).
Nonspecific binding was determined with 1 µM WF-23, a potent tropane
analog with <0.1 nM Ki values at
dopamine and 5-HT transporters (Bennett et al., 1995). For both
[125I]RTI-55 and
[3H]PTT binding in rat striatal membranes, 1 µM WF-23 provided the same level of nonspecific binding as 30 µM
cocaine or 5 µM mazindol. All assays were performed in triplicate,
with less than 5% S.D. between replicate samples. Data are expressed
as mean values ± S.E. of at least three separate experiments.
IC50 values were calculated by iterative
nonlinear regression of concentration-effect curves (JMP; SAS
Institute, Cary, NC) prepared with at least six concentrations of
unlabeled compound. KD and
Bmax values were calculated from
saturation analyses using one- and two-site fits by computer analysis
using LIGAND (Munson and Rodbard, 1980). Ki values were determined from
IC50 values using the Cheng-Prusoff relationship
(Cheng and Prusoff, 1973).
High-Affinity Dopamine Uptake.
[3H]Dopamine uptake was determined in crude
synaptosomal preparations from rat striatum as described previously
(Bennett et al., 1995). Briefly, striatal P2
pellets (0.5 mg protein/ml), prepared from a 48,000g
centrifugation of a postnuclear (3000g) supernatant, were
suspended in a total volume of 250 µl of assay buffer, preincubated
with unlabeled drugs for 10 min at 37°C, then incubated with 15 nM
[3H]dopamine for 20 min at 37°C. Uptake was
terminated by the addition of ice-cold assay buffer and filtration
through Whatman GF/B filters. Nonspecific uptake was determined in the
presence of 10 µM mazindol. All assays were performed in triplicate,
and data are expressed as mean values ± S.E. of at least three
separate experiments.
In Vitro Autoradiography.
For the initial characterization
of [3H]PTT binding in rat brain, male
Sprague-Dawley rats (Harlan) were sacrificed by an overdose of sodium
pentobarbital; brains were removed and quick-frozen in isopentane
cooled over dry ice to 40°C. Brains were stored at 80°C.
Twenty-micrometer sections were cut at 20°C and thaw-mounted onto
chrome alum/gelatin-subbed slides, desiccated, and frozen at 80°C
until processing.
Optimal conditions for autoradiography were determined in coronal
sections through the caudate-putamen. Experiments were conducted first
to establish optimal times for association of
[3H]PTT at the dopamine transporter. Tissue
sections were preincubated at 4°C in Tris-NaCl buffer (50 mM
Tris-HCl, pH 7.4, 100 mM NaCl) for 10 min to remove any endogenous
dopamine. Sections were then incubated at room temperature with
[3H]PTT (2.5 nM) for various times ranging from
0 to 60 min. After a 30-s rinse in distilled water, sections were
swiped from slides with a glass fiber filter (GF/C, 2.4 cm; Whatman),
transferred to scintillation vials, and incubated overnight in
scintillation fluid for determination of radioactivity by liquid
scintillation spectrophotometry. Nonspecific binding was determined in
adjacent sections in the presence of 2.5 µM WF-23 (Bennett et al.,
1995). To establish optimal times for washout of bound ligand from
tissue, tissue sections were incubated as above in Tris-NaCl buffer
with [3H]PTT (2.5 nM) alone or in combination
with 2.5 µM WF-23 at room temperature for 0 to 30 min. Radioactivity
was determined as described above.
Saturation binding studies were conducted with the standard protocol
described above to estimate the KD
value of [3H]PTT. The concentration of
radioligand was varied from 0.01 to 50 nM. Nonspecific binding was
defined with WF-23 as above. The KD
and Bmax values were calculated using
nonlinear regression analysis with LIGAND (Munson and Rodbard, 1980).
To characterize the distribution of
[3H]PTT-binding sites in brain, coronal
sections were collected at regular intervals throughout the
rostral-caudal extent of the rat brain. The slide-mounted tissue was
preincubated for 10 min at 0-4°C in Tris-NaCl buffer. Tissue
was then incubated for 40 min with 5 nM [3H]PTT
in Tris-NaCl buffer at room temperature to determine specific binding,
whereas adjacent sections were incubated with 5 nM
[3H]PTT in the presence of 5 µM WF-23
to determine nonspecific binding. All sections were rinsed for 5 min in
fresh Tris-NaCl buffer at 0-4°C, followed by a 10-s rinse in
distilled water at 0-4°C to remove excess salts. Slides were dried
under a stream of cool air, then opposed to film (tritium-sensitive
Hyperfilm 3H; Amersham) for 5 days in the
presence of tritium standards. After exposure, films were developed
with Kodak GBX developer, fixed, and rinsed.
The visualization of the distribution of
[125I]RTI-55-binding sites was conducted
according to procedures adapted from Boja et al. (1992). Tissue was
prepared as described above, and slide-mounted sections were
preincubated for 10 min in sodium phosphate buffer containing 0.32 M
sucrose, pH 7.4, at 24°C. Tissue was then incubated for 120 min at
23°C with 0.05 nM [125I]RTI-55 in the above
buffer to determine specific binding. Nonspecific binding was defined
using 50 µM ()-cocaine. All sections were rinsed for 5 min in
sodium phosphate buffer at 0-4°C, followed by a 10-s rinse in
distilled water at 0-4°C to remove excess salts. Slides were dried
under a stream of cool air, then opposed to Hyperfilm
3H for 5 days in the presence of
3H standards. After exposure, films were
developed with Kodak GBX developer, fixed, and rinsed.
Analysis of autoradiograms was conducted by computerized quantitative
densitometry. Tissue equivalent values (femtomoles per milligram of wet
weight tissue) were determined from the optical densities and from a
calibration curve obtained by densitometric analysis of the
autoradiograms of tritium standards. Specific binding was determined by
digitally subtracting nonspecific binding from the total binding, as
measured in adjacent sections.
6-Hydroxydopamine (6-OHDA) Lesion Studies.
Male
Sprague-Dawley rats were pretreated i.p. with 25 mg/kg desipramine
hydrochloride 40 to 60 min before lesions. A 3.6-µg/ml solution of
6-OHDA hydrobromide (Research Biochemicals, Natick, MA) in saline with
0.2 mg/ml L-(+)-ascorbic acid (J.T. Baker Inc., Phillipsburg, NJ) was prepared immediately before use. Unilateral (right hemisphere) infusions of 3.5 and 2.5 µl of 6-OHDA were delivered to a medial and lateral substantia nigra pars compacta site,
respectively, with the medial coordinate aimed at ablating ventral
tegmentum area as well as substantia nigra pars compacta. Injections were delivered at 0.5 µg/min, with the needle bevel directed caudally for both injection sites. Rats were sacrificed as
described above 14 days after the lesion. Coronal sections throughout
the rostral-caudal extent of the forebrain were collected as described
above and stored at 80°C until processing.
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Results |
Structure and Pharmacology of Tropane Analogs.
The structures
of several tropanes are compared with cocaine in Fig.
1. Both RTI-55 and PTT lack the benzyl
ester moiety of cocaine, and both contain para substituents
on the aryl ring (methyl in the case of PTT; iodine in the case of
RTI-55). One significant difference between PTT and RTI-55 is at the
2-position of the tropane ring, where RTI-55 has a methyl ester and PTT
has an ethyl ketone. Two other relevant tropane structures in Fig. 1
are WF-23, an extremely potent compound that binds to both dopamine and
serotonin transporters with similar affinities, and WF-31, a compound
that is relatively selective in binding to serotonin transporters
(Bennett et al., 1995).
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Fig. 1.
Structures of selected tropane analogs compared with
cocaine and RTI-55. PTT is relatively selective at dopamine
transporters; the naphthyl analog WF-23 is highly potent and
nonselective at dopamine, 5-HT, and norepinephrine transporters; and
the isopropyl-phenyl derivatives WF-31 and WF-60 are selective in
binding to 5-HT transporters.
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The pharmacology of these compounds at biogenic amine transporters, as
revealed by displacement of selective radioligands and uptake studies,
reveals that cocaine and [125I]RTI-55 are
relatively nonselective for dopamine and 5-HT transporters (Table
1). In transporter binding and uptake
studies, PTT is 20 to 140 times more potent in binding to dopamine
transporters than to 5-HT transporters (Table 1). To prepare this
radioligand, the N-demethylated analog of PTT was reacted
with [3H]methyl iodide to produce
[3H]PTT, which was then used in binding studies
in both brain membranes and sections.
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TABLE 1
Potencies of tropanes at biogenic amine transporters
Values listed are from Bennett et al. (1995) and Boja et al. (1991).
Values for PTT refer to the racemic
mixture.
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Binding Parameters of [3H]PTT in Striatal
Membranes.
The kinetics of [3H]PTT binding
to rat striatal membranes are shown in Fig.
2. [3H]PTT (1 nM)
achieved equilibrium binding within 20 min (Fig. 2A), with an estimated
half-time of association of 3.5 min. Dissociation rate was determined
by addition of excess (1 µM) unlabeled PTT after a 30-min incubation
of [3H]PTT in rat striatal membranes. Results
(Fig. 2B) revealed that [3H]PTT binding was
readily reversible, with a t1/2 of 4 min. This rate of dissociation was somewhat faster than that of
[125I]RTI-55, which exhibited a
t1/2 of 8 min (not shown).
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Fig. 2.
Kinetics of [3H]PTT binding in rat
striatal membranes. A, association: [3H]PTT was incubated
with striatal membranes from 0 to 40 min before terminating the
reaction by filtration. Data are expressed as distintegrations per
minute [3H]PTT bound per microgram of protein. B,
dissociation: after a 30-min preincubation of [3H]PTT
with striatal membranes, 1 µM unlabeled PTT was added and the
reactions were terminated at various times. Data are expressed as
percentage of [3H]PTT bound with no added unlabeled
PTT.
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Equilibrium binding parameters were determined for
[3H]PTT and
[125I]RTI-55 by incubating various
concentrations of the radioligands with striatal membranes for 30 min.
Scatchard analysis of [3H]PTT binding (Fig.
3A) revealed binding that was best fit to a single site, with a KD value of
5.1 ± 0.4 nM and a Bmax value of
0.33 ± 0.12 pmol/mg protein. In contrast, Scatchard analysis of
[125I]RTI-55 binding (Fig. 3B) was best fit to
a biphasic model. For the high-affinity site, the
KD value was 0.045 ± 0.020 nM,
and the Bmax value was 0.23 ± 0.069 pmol/mg. For the low-affinity [125I]RTI-55 site, the
KD value was 3.1 ± 0.55 nM and
the Bmax value was 8.6 ± 1.2 pmol/mg. These values for [125I]RTI-55 binding
agree closely with those reported previously, with a high-affinity
binding KD value of 0.11 nM and a
Bmax value of 0.16 pmol/mg; a
low-affinity KD value of 2.57 nM and a
Bmax value of 0.57 pmol/mg (Boja et
al., 1991). The Bmax value for the
single class of [3H]PTT binding was not
significantly different from that of the high-affinity site for
[125I]RTI-55 binding (P = .89, Student's t test) but was significantly different from the
low-affinity KD value of
[125I]RTI-55 binding (P < .001).
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Fig. 3.
Scatchard plots of [3H]PTT binding (A)
and [125I]RTI-55 binding (B) in rat striatal membranes.
Data are typical plots from experiments that were repeated at least
three times. Lines represent best fit parameters as determined for
single-site analysis (for [3H]PTT) and two-site analysis
(for [125I]RTI-55) by LIGAND.
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To determine the overall pharmacology of
[3H]PTT binding, displacement by a number of
biogenic amine transport inhibitors was determined against both
[3H]PTT and
[125I]RTI-55 binding, as well as in
[3H]dopamine uptake assays, in rat striatal
preparations. Typical concentration-effect curves for several compounds
in displacing [3H]PTT binding are presented in
Fig. 4, including fluoxetine, GBR 12909, cocaine, and unlabeled PTT. All compounds inhibited 100% of specific
[3H]PTT binding with various degrees of
potency. As predicted for a dopamine transporter radioligand, GBR 12909 was most potent, followed by PTT itself, cocaine, and fluoxetine.
Analysis of competition assays (Table 2)
showed that the rank order of potencies of compounds in displacing
[3H]PTT and
[125I]RTI-55 binding generally correlated with
one another. For example, the most potent analog tested against both
[3H]PTT and
[125I]RTI-55 was the 2-naphthyl tropane analog
WF-23. Moreover, the binding of [3H]PTT was
relatively dopamine-selective, with the selective 5-HT analogs WF-31,
paroxetine, and fluoxetine providing relatively low potencies in
displacing [3H]PTT binding. Finally, the rank
order of these compounds in displacing [3H]PTT
binding were the same as that in inhibiting
[3H]dopamine uptake.
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Fig. 4.
Concentration-effect curves of GBR 12909 ( ),
unlabeled PTT ( ), cocaine ( ), and fluoxetine ( ) in displacing
[3H]PTT binding to rat striatal membranes.
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TABLE 2
Ki and IC50 values of uptake
inhibitors in displacing [125I]RTI-55 binding and
[3H]PTT binding and in inhibiting
[3H]dopamine uptake in rat striatum
Ki, IC50, and Hill slope
(nH) values were determined for the selected
drugs in displacing [125I]RTI-55 and
[3H]PTT binding in rat striatal membranes and in
inhibiting [3H]dopamine uptake in striatal synaptosomes,
as described in Experimental Procedures.
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Nevertheless, there were several interesting differences between
several compounds in displacing [3H]PTT binding
compared with [125I]RTI-55 binding. For many
compounds, Hill slopes in displacing [3H]PTT
binding were higher than those in displacing
[125I]RTI-55 binding (for example, see
desipramine, GBR compounds, and all 5-HT-selective analogs in Table 2).
In general, the potencies of dopamine-selective compounds were similar
between the two binding assays; however, significant differences in
potencies were observed for several nonselective compounds (e.g.,
cocaine, WF-23, and desipramine), as well as all of the 5-HT-selective
compounds (WF-31, fluoxetine, paroxetine, and WF-60). In all cases of
these discrepancies, the 5-HT-selective compounds were significantly
more potent in displacing [125I]RTI-55 binding
than [3H]PTT binding in the same striatal
membranes. To confirm these differences, the potencies of these
compounds in displacing both [125I]RTI-55 and
[3H]PTT binding were compared with their
IC50 values in inhibiting [3H]dopamine uptake in striatal synaptosomes
(Table 2). These results showed that the potencies of several compounds
in inhibiting [3H]dopamine uptake were closer
to those in displacing [3H]PTT binding than in
displacing [125I]RTI-55 binding. These
relationships are demonstrated in the correlation plots in Fig.
5, which correlate
IC50 and Ki
values for these transporter inhibitors in all three assays. All
correlations were significant, including dopamine uptake versus
[3H]PTT binding (Fig. 5A, r = 0.95), dopamine uptake versus [125I]RTI-55
binding (Fig. 5B, r = 0.89), and
[125I] RTI-55 binding versus
[3H]PTT binding (Fig. 5C, r = 0.94). However, within each correlation, interesting differences can be
seen. For drugs that were potent in binding to dopamine transporters
(log Ki < 2), the correlation with
dopamine uptake IC50 values was generally
excellent whether the radioligand was
[125I]RTI-55 or
[3H]PTT. However, less significant correlation
between [125I]RTI-55 binding and dopamine
uptake was observed with drugs that were less potent in binding to
dopamine transporters (log Ki > 2),
many of which were 5-HT-selective, NE-selective, and nonselective transporter inhibitors. When correlations were plotted for only those
drugs with log Ki values >2 (not
shown), then the differences in correlations were much more pronounced:
r = 0.92 for dopamine uptake versus
[3H]PTT, r = 0.45 for dopamine
uptake versus [125I] RTI-55, and
r = 0.64 for [125I]RTI-55
versus [3H]PTT.
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Fig. 5.
Correlations of potencies of biogenic amine
transporter inhibitors versus [3H]PTT binding,
[125I]RTI-55 binding, and [3H]dopamine
uptake. A, dopamine uptake versus [3H]PTT binding; B,
dopamine uptake versus [125I]RTI-55 binding; C,
[125I]RTI-55 binding versus [3H]PTT
binding. Data are expressed as logarithms of
Ki values (for the two binding assays) and
IC50 values for dopamine uptake and based on values
obtained from Table 2.
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In Vitro Autoradiography.
Association, washout, and saturation
experiments were conducted in tissue sections to determine optimal
parameters for autoradiography. Association of
[3H]PTT in tissue sections was rapid, and
specific binding reached equilibrium within 30 to 40 min (Fig.
6A), remaining stable thereafter. In
washout experiments, in unwashed tissue (0 time), total and nonspecific
binding were essentially equivalent (see Fig. 6B). Washout of excess
[3H]PTT in tissue sections occurred with wash
time of 5 min. Washout of nonspecific binding also occurred rapidly and
reached minimal levels by 5 min. At this time point, nonspecific
binding constituted <15% of the total binding. Longer wash times did
not increase the proportion of specific binding. From these data, a
rinse time of 5 min was chosen for subsequent studies.
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Fig. 6.
Kinetics of [3H]PTT binding in rat
tissue sections. A, association: slide-mounted tissue sections were
incubated with 2.5 nM [3H]PTT alone (total) or in
combination with 2.5 µM WF-23 (nonspecific) for various times ranging
from 0 to 60 min. After incubation, sections were rinsed for 30 s,
then wiped from the slide. B, washout: tissue sections were incubated
for 40 min with 2.5 nM [3H]PTT alone or in combination
with 2.5 µM WF-23 to achieve equilibrium binding. To establish the
time required for dissociation of excess [3H]PTT, the
sections were immersed in fresh buffer for various times ranging from 0 to 30 min, then wiped from slides.
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In saturation experiments in brain sections, specific
[3H]PTT binding approached saturation at 40 nM,
whereas nonspecific binding increased linearly with increasing
radioligand concentrations (Fig. 7A).
Scatchard analysis of saturation data (Fig. 7B) revealed specific
binding that was best fit to a single site, with a
KD value of 18 nM in tissue sections.
Moreover, the Hill slope of the [3H]PTT-binding
data was 0.99, yielding additional evidence of a single binding site.
The final parameters for autoradiographic analysis of slide-mounted
tissue sections, chosen on the basis of the experiments described
above, were preincubation for 10 min, incubation for 40 min with 5 nM
[3H]PTT, followed by a 5-min rinse in buffer
and a 10-s rinse in water. Under these conditions, the optimal film
exposure time was determined to be 5 days, and film optical density
approached saturation by 7 days. Nonspecific binding was not
perceptible above background levels (i.e., specific binding >95% of
total binding) (Fig. 8).
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Fig. 7.
Saturation analysis of [3H]PTT binding
to brain sections. A, slide-mounted sections were incubated in one of
eight concentrations of [3H]PTT (0.05-25 nM) alone or in
the presence of WF-23 (0.05-25 µM). Slides were incubated for 40 min, then rinsed in fresh buffer for 5 min, and tissue-bound
radioactivity was wiped from the slides. B, Scatchard analysis of
[3H]PTT-binding data. Data are from experiments that were
conducted on tissue from three different brains. Line represents best
fit parameters as determined for single-site analysis by LIGAND.
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Fig. 8.
Autoradiograms of [3H]PTT binding in
coronal rat brain sections at various levels. A, total
[3H]PTT binding at the level of the anterior caudate and
nucleus accumbens. B, nonspecific binding, as defined by the presence
of excess WF-23 in an adjacent section. Nonspecific binding was less
than 95% of total binding. C, total [3H]PTT binding at
the level of the middle caudate, where the nucleus core and shell are
well differentiated. D, total [3H]PTT binding at the
level of the posterior caudate. E, total [3H]PTT binding
at the level of the substantia nigra and ventral tegmental area. Note
the low binding levels in the hippocampus. F, unilateral 6-OHDA lesions
of the substantia nigra/ventral tegmental area ablated
[3H]PTT binding in the caudate, nucleus accumbens,
olfactory tubercle, and cingulate cortex.
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The distribution of [3H]PTT-binding sites was
distinctly heterogeneous throughout rat brain. The highest densities of
[3H]PTT-binding sites (>175 fmol/mg tissue)
were found throughout the caudate nucleus, core of the nucleus
accumbens, and the olfactory tubercle (Table 3).
Moderate levels of binding (75-175 fmol/mg tissue) were present in the
shell of the nucleus accumbens and the ventral midbrain area, whereas
lower levels of binding (35-75 fmol/mg tissue) were observed in the
median forebrain bundle, locus ceruleus, dorsal raphe, substantia nigra
pars reticulata, substantia nigra pars lateralis, and caudal linear
nucleus (Table 3). The following regions contained detectable, but low
levels of binding (30 fmol/mg tissue or less): anterior cingulate
cortex, medial prefrontal cortex, lateral septum, globus pallidus, bed nucleus stria terminalis, paraventricular thalamus, antero-ventral thalamus, supraoptic nucleus, medial-dorsal thalamus, periventricular hypothalamus, lateral habenula, portions of the amygdala, zona incerta,
hippocampus, superior colliculus, and central gray.
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TABLE 3
Regional distribution of [3H]PTT-binding sites in rat
brain
[3H]PTT binding was performed on coronal rat brain
sections as described in Experimental Procedures. Films were
analyzed for optical density after exposure to tissue sections for 5 days. Data are expressed as femtomoles of 3H per milligram
of wet weight tissue ± S.E.M.
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The highest density of [3H]PTT-binding sites
was evident throughout the rostral-caudal extent of the dorsal and
ventral striatum. To ensure that
[3H]PTT-binding sites were in fact
dopaminergic, a series of lesions was performed. The dopaminergic
neurotoxin, 6-OHDA, was stereotaxically injected unilaterally into the
substantia nigra/ventral tegmental area of rats. This resulted in a
profound reduction (>95% depletion compared with the contralateral
side) of [3H]PTT binding in the ipsilateral
caudate and nucleus accumbens (Fig. 8). Binding was also abolished in
olfactory tubercle, anterior cingulate cortex, medial prefrontal
cortex, amygdala, and median forebrain bundle ipsilateral to the side
of the lesion. Destruction of dopaminergic cell bodies by the
neurotoxin, therefore, eliminated binding sites on terminals of the
mesostriatal, mesocortical, and mesolimbic dopaminergic pathways.
The pattern of [3H]PTT binding was similar to
the brain areas labeled by [125I]RTI-55 (see
Table 4). Similar binding was seen in
dopaminergically innervated areas, including caudate nucleus, nucleus
accumbens, and ventral midbrain. Both the brain areas labeled and the
intensity of binding were largely similar within these regions. In
other brain regions such as hippocampus, lateral geniculate, medial hypothalamus, and anterior thalamus (i.e., areas with substantial serotonergic innervation), labeling of
[125I]RTI-55-binding sites was more intense
than with [3H]PTT. These data are consistent
with the lack of specificity of [125I]RTI-55
compared with [3H]PTT at dopamine transporters.
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TABLE 4
Comparison of regional distribution of [3H]PTT
binding with [125I]RTI-55 binding in rat brain
Autoradiography of [3H]PTT and [125I]
RTI-55 binding was performed as described in Experimental
Procedures. Data are expressed as relative optical densities, with
the caudate nucleus assigned a value of
10.
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To test the possibility that the moderate
[3H]PTT binding in locus ceruleus was caused by
binding to norepinephrine, [3H]PTT binding was
measured in sections from dorsal and ventral striatum, neocortex, and
locus ceruleus, with various concentrations (1, 10, and 100 nM) of the
norepinephrine transporter inhibitor nisoxetine (data not shown).
Although the lowest concentration of nisoxetine had no effect on
binding levels in any region, the higher concentrations of nisoxetine
eliminated [3H]PTT binding in locus ceruleus,
with no effect on [3H]PTT binding in any other
brain region measured. This suggests that the presence of
[3H]PTT binding in locus ceruleus is due to
labeling of norepinephrine transporters.
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Discussion |
The results of this study confirm the utility of the novel
dopamine transporter-selective tropane [3H]PTT
in binding studies in both striatal membranes and in brain sections.
The binding of [3H]PTT to rat striatal
membranes is rapid, reversible, has high affinity for DAT, and contains
a low level (<5%) of nonspecific binding. The membrane-binding data
confirm that the binding of [3H]PTT corresponds
closely to that previously determined for the unlabeled form of PTT. In
particular, the KD value of
[3H]PTT binding (5 nM) is similar to the
Ki value of the active enantiomer of
PTT in displacing [125I]RTI-55 binding (3.5 nM). Moreover, the pharmacological specificity of
[3H]PTT, with dopamine transporter-selective
compounds being more potent in displacing binding than 5-HT-selective
compounds, is also consistent with previous results obtained both in
vitro (Davies et al., 1993, 1994) and in vivo (Porrino et al., 1994).
However, the kinetics of [3H]PTT binding to
striatal membranes, with a half-time of dissociation of 4 to 5 min, is
not consistent with the in vivo effects of PTT. When administered by
single i.p. injections, PTT increased locomotor activity in rats for up
to 3 to 4 h (Porrino et al., 1994, 1995), and a similar time
course was observed in its ability to increase extracellular dopamine levels in nucleus accumbens by microdialysis (Hemby et al., 1995). Although these long durations of action are observed for other high-affinity tropanes as well (Reith et al., 1986; Cline et al., 1992), the results of this study clearly demonstrate that these in vivo
time courses cannot be explained on the basis of the intrinsic dissociation rate of the tropane from the dopamine transporter.
Although both [125I]RTI-55 and
[3H]PTT binding share several common
properties, there are several important differences. The most obvious
difference in membrane-binding data is in the relative affinities of
the two radioligands, with a high-affinity
KD value of 0.05 nM for
[125I]RTI-55, compared with 5 nM
KD value for
[3H]PTT binding. Moreover, although a
low-affinity (KD value of 3 nM)
component was clearly evident in [125I]RTI-55
binding, as reported for several tropane radioligands (Madras et al.,
1989b; Boja et al., 1991), the binding of
[3H]PTT was best fit to a single-site model. In
addition, comparisons of several compounds in displacing binding (Table
2) revealed lower Hill slopes versus
[125I]RTI-55 binding than versus
[3H]PTT binding. It is not likely that the lack
of a low-affinity site was due to the fact that the
[3H]PTT saturation analyses did not use high
enough concentrations of PTT to observe such a site because homologous
displacement of [3H]PTT binding by
concentrations of up to 1 µM PTT showed a Hill slope of 0.9 and,
therefore, no evidence of multiple binding sites. Nevertheless, despite
these differences between the forms of the saturation plots for these
two tropane radioligands, the Bmax value of [3H]PTT binding (0.33 pmol/mg) was
similar to the Bmax value of the
high-affinity [125I]RTI-55 site (0.23 pmol/mg).
The appearance of multiple binding sites for tropane radioligands is
not a universal finding: Reith et al. (1992) have reported only one
site for [3H]CFT binding in brain membranes,
and Rothman et al. (1994) have reported a single site for
[125I]RTI-55 binding in caudate membranes and
in transfected COS cells. Nevertheless, in this study we found clear
evidence of multiple sites for [125I]RTI-55
binding in striatal membranes, and the difference between these sites
and the single-site binding of [3H]PTT was evident.
Another important difference between [3H]PTT
and [125I]RTI-55 binding in striatal membranes
involved the pharmacology of the two ligands. Although compounds that
were relatively selective in binding to dopamine transporters displayed
approximately the same potencies in displacing both radioligands,
nonselective and 5-HT-selective compounds were significantly less
potent in displacing [3H]PTT binding than
[125I]RTI-55 binding. The result of these
differences was to provide a lower level of correlation between
dopamine uptake and [125I]RTI-55 binding than
between uptake and [3H]PTT binding (Fig. 5),
especially with 5-HT-selective compounds. It is well known that
[125I]RTI-55 is relatively nonselective in
binding both dopamine and 5-HT transporters (Boja et al., 1991) and
that the principal utility of using
[125I]RTI-55 in binding to dopamine
transporters in striatal membranes is the fact that relatively few 5-HT
transporters exist in rat striatum compared with the extraordinarily
high levels of dopamine transporters. The results of this study suggest
that enough 5-HT transporters exist in these striatal membranes to
provide an artificially high potency of 5-HT-selective compounds in
[125I]RTI-55-binding assays (Rothman et al.,
1994). The differences in potencies of even the most selective 5-HT
transporter inhibitors between [125I]RTI-55 and
[3H]PTT binding is not large, but this
difference is sufficient to explain most of the discrepancies between
[125I]RTI-55-binding data and those from
[3H]dopamine uptake experiments (Bennett et
al., 1995).
The characteristics of [3H]PTT binding in
tissue sections were similar to that of [3H]PTT
binding in striatal homogenates. For example,
[3H]PTT exhibited single-site binding
characteristics in tissue sections similar to its binding in
homogenates. The estimated KD value of
[3H]PTT in tissue sections, however, was 18 nM,
higher than the 5 nM KD value
determined in tissue homogenates. Such differences are not uncommon
when determinations are made autoradiographically in tissue sections.
An incubation concentration of 5 nM was chosen for the autoradiographic
studies because higher concentrations were associated with higher
levels of nonspecific binding.
The distribution of [3H]PTT-binding sites was
distinctly heterogeneous with the highest levels of binding present in
the caudate-putamen and nucleus accumbens, regions known to be rich in
dopamine transporters. Moderate levels of binding were also found in
the substantia nigra and ventral tegmental area within the midbrain, as
well as in portions of the hypothalamus and the anterior cingulate
cortex. Binding in other brain regions, including neocortex, globus
pallidus, thalamus, substantia nigra reticulata, and hippocampus, was
just above background levels. This distribution is similar to that seen
with other radioligands that have been shown to bind to the dopamine
transporter including [3H]WIN 35,428 (Madras et
al., 1989b; Canfield et al., 1990),
[125I]RTI-55 (Boja et al. 1991),
[125I]RTI-121 (Boja et al., 1992),
[3H]GBR12909 (Richfield, 1991), and
[3H]mazindol (Javitch et al., 1985). The
distribution of [3H]PTT-binding sites in tissue
sections was also consistent with the immunochemical distribution of
the dopamine transporter determined with immunogold (Ciliax et al.,
1995; Nirenberg et al., 1997), as well as the topography of
dopaminergic innervation observed in histofluorescence studies
(Björklund and Lindvall, 1984). Furthermore, unilateral
destruction of dopaminergic cells in the ventral midbrain with the
selective neurotoxin 6-OHDA completely abolished
[3H]PTT binding on the side of the lesion. For
example, [3H]PTT binding in the caudate and
nucleus accumbens was less than 5% of binding levels on the intact
side. These findings also support the specificity of
[3H]PTT binding for the dopamine transporter.
The advantages of [3H]PTT, however, include its
binding to a single site, very low levels of nonspecific binding, and
the short exposure times necessary for film autoradiography.
As in membrane preparations, there were clear differences between the
distribution of [3H]PTT-binding sites and
binding sites defined with [125I]RTI-55. The
present data and previous reports have shown significant levels of
[125I]RTI-55 binding in thalamus, cerebral
cortex, and substantia nigra reticulata, areas known to have few
dopaminergic transporters but to be rich in serotonergic transporters.
Given that PTT is 20 to 140 times more potent at the dopamine
transporter as compared with the serotonin transporter, it is possible
that significant binding might be present in regions with high levels
of serotonin transporters. With the exception of the raphe, however,
[3H]PTT binding was very low in
serotonergically innervated areas. The short exposure times necessary
for [3H]PTT undoubtedly contribute to the low
levels of detectable binding in these areas as compared with regions
rich in the dopamine transporter. Moderate levels of binding were also
present in the raphe nuclei and the locus ceruleus, which contain
serotonin and norepinephrine cell bodies, respectively. Furthermore,
because dopaminergic afferents to the raphe nuclei have been well
documented (Beckstead et al., 1979; Simon et al., 1979; Marchand and
Hagino, 1983), dopamine transporters on these projections may also
contribute to the levels of detectable [3H]PTT
binding in this area. The presence of [3H]PTT
binding in locus ceruleus, however, cannot be explained on the basis of
binding to dopamine transporters. Because PTT demonstrates some
affinity for the norepinephrine transporter (Bennett et al., 1995) and
locus ceruleus contains high levels of norepinephrine transporters, the
presence of detectable labeling in the locus ceruleus is not
surprising. The blockade of [3H]PTT binding in
locus ceruleus by nisoxetine confirmed this possibility. The absence of
any effects of nisoxetine on [3H]PTT binding in
other brain areas also indicates that the contribution of binding to
the norepinephrine transporter in these areas is minimal. Moreover, the
overall distribution of [3H]PTT did not
correspond to that of the norepinephrine transporter ligand,
[3H]nisoxetine (Tejani-Butt, 1991), because no
[3H]PTT binding was evident in the hippocampus
or cerebellar cortex.
[3H]PTT demonstrated rapid film exposure times,
on the order of 5 days, compared with 4 to 7 weeks for most tritiated
dopamine transporter ligands (Javitch et al., 1985; Canfield et al.,
1990). Thus, [3H]PTT offers the advantages of
short film exposure time, which is available with
125I-labeled ligands (Boja et al., 1992) without
the decreased neuroanatomical resolution of
125I-labeled compounds. It is unclear at this
point why [3H]PTT autoradiography develops so
quickly because the specific activity of
[3H]PTT is similar to other tritiated ligands
(Canfield et al., 1990). It is possible that the relatively high
affinity of PTT for dopamine transporters contributes to this short
exposure time because the relatively low concentrations of radioligand
that are used in these experiments would occupy a higher percentage of
transporter sites than those of a lower affinity radioligand. However,
the affinity of [3H]PTT is not very different
from that of [3H]CFT (Canfield et al., 1990),
which has a longer film exposure time than
[3H]PTT. So differences in transporter
affinities cannot be the full explanation of these differences.
In summary, [3H]PTT has high affinity and
selectivity for dopamine transporters and exhibits single-site binding
characteristics in both striatal homogenates and tissue sections. In
addition, [3H]PTT has extremely fast film
exposure times and low nonspecific binding. These properties of
[3H]PTT indicate that it will be a superior
ligand for autoradiographic localization of the dopamine transporter.
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Abstract
Introduction
