Lobelane analogues containing 4-hydroxy and 4-(2-fluoroethoxy) aromatic substituents: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

Article abstract of DOI:10.1016/j.bmcl.2016.03.119

A series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and 4-(2-fluoroethoxy) substituents were synthesized and evaluated for inhibition of [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and th

Full text of DOI:10.1016/j.bmcl.2016.03.119

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2422–2427
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lobelane analogues containing 4-hydroxy and 4-(2-fluoroethoxy)
aromatic substituents: Potent and selective inhibitors of
[3H]dopamine uptake at the vesicular monoamine transporter-2
Shyamsunder R. Joolakanti a,y, Justin R. Nickell b,y, Venumadhav Janganati a, Guangrong Zheng a,
Linda P. Dwoskin b, Peter A. Crooks a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and 4-(2-fluoroethoxy)
substituents were synthesized and evaluated for inhibition of [3H]dopamine (DA) uptake at the vesicular
monoamine transporter-2 (VMAT2) and the dopamine transporter (DAT), and [3H]serotonin uptake at the
serotonin transporter (SERT). Most of these compounds exhibited potent inhibition of DA uptake at
VMAT2 in the nanomolar range (Ki = 30–70 nM). The two most potent analogues, 7 and 14, both exhibited
a Ki value of 31 nM for inhibition of VMAT2. The lobelane analogue 14, incorporating 4-(2-fluoroethoxy)
and 4-hydroxy aromatic substituents, exhibited 96- and 335-fold greater selectivity for VMAT2 versus
DAT and SERT, respectively, in comparison to lobelane. Thus, lobelane analogues bearing hydroxyl and
fluoroethoxy moieties retain the high affinity for VMAT2 of the parent compound, while enhancing
selectivity for VMAT2 versus the plasmalemma transporters.
Received 10 March 2016
Revised 30 March 2016
Accepted 31 March 2016
Available online 1 April 2016
Keywords:
4-Hydroxyphenyl lobelane analogues
Fluorinated lobelane analogues
Vesicular monoamine transporter-2
Dopamine uptake
Ó 2016 Elsevier Ltd. All rights reserved.
Dopamine and serotonin transporter
Methamphetamine (METH) abuse is a serious burden on the
United States, with approximately 100,000 new METH users every
year.1 METH abuse constitutes significant health risks, such as long
term neuronal damage, psychosis, paranoia, insomnia, anxiety,
aggression, delusions, and hallucinations, and chronic use ulti-
mately may lead to death.2 Currently, there are no FDA-approved
medications to treat METH addiction. METH users describe a
sudden rush of pleasure lasting for several minutes to hours
upon self-administration of the drug. These reinforcing properties
of METH result from METH-induced prolonged release of
dopamine (DA) into the extracellular space, where it interacts
with postsynaptic DA receptors.3 METH enters dopaminergic
presynaptic terminals via passive diffusion through the plasma-
lemma membrane, and as a substrate for the plasmalemma DA
transporter (DAT).4 Once inside the terminal, METH evokes the
release of vesicular DA from synaptic vesicles into the cytosol
through an interaction with the vesicular monoamine trans-
porter-2 (VMAT2).4 Since METH also inhibits the activity of the
mitochondrial enzyme, monoamine oxidase (MAO), the METH-
evoked increase in cytosolic DA is not subjected to metabolism,
and the formation of dihydroxyphenylacetic acid (DOPAC).5 The
increase in cytosolic DA concentrations makes DA more readily
available for release into the extracellular compartment via rever-
sal of DAT. The resulting release of DA from the presynaptic termi-
nal, and the subsequent enhanced stimulation of postsynaptic DA
receptors results in the reinforcing effects and the high abuse
liability associated with METH.6 In this respect, heterologous VMAT2
knockout mice exhibit reduced amphetamine conditioned reward,
enhanced amphetamine locomotion, and enhanced sensitivity to
amphetamine, clearly indicating the importance of VMAT2 in
mediating the behavioral effects of this related psychostimulant.7
Lobeline is the principal alkaloid of the Indian tobacco plant,
Lobelia inflata. Lobeline inhibits DA uptake into synaptic vesicles
via an interaction with the tetrabenazine (TBZ) binding site on
VMAT2, but does not inhibit MAO activity.8 Lobeline attenuates
METH self-administration in a rodent model, but importantly, does
not substitute for METH in the self-administration paradigm, indi-
cating that it lacks abuse liability.9 Lobeline also decreases METH-
induced hyperactivity and behavioral sensitization in rats.9 Also,
lobeline decreases METH-evoked DA release from superfused rat
striatal slices, while concurrently increasing extracellular DOPAC.8a
Based on these preclinical findings, lobeline was considered a lead
candidate as a treatment for METH abuse. Unfortunately, lobeline
also acts as a nicotinic acetylcholine receptor antagonist.10 Drugs
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
y
These authors contributed equally to this manuscript and are co-first authors.
http://dx.doi.org/10.1016/j.bmcl.2016.03.119
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

S. R. Joolakanti et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2422–2427
2423
modification significantly improved affinity for VMAT2 and
drug-likeness properties.12 Notably, this compound blocked METH
self-administration in rats, but had no effect on responding for food.13
In the present study, we report on the synthesis of several new
lobelane derivatives that incorporate novel functional groups onto
the phenyl rings of lobelane and GZ-793A.
OH
O
N
CH3
Lobeline
N
H
Nor-Lobelane
Moreover, several of the synthesized compounds were fluorine-
containing analogues and are of potential value in positron
emission tomography (PET) studies when prepared incorporating
the 18F positron-emitting isotope (half-life ꢀ120 min). The
incorporation of fluorine into the structure of a drug molecule
also influences lipophilic, electronic, and metabolic properties, all
of which can critically influence both the pharmacodynamic and
pharmacokinetic properties of the drug.14
N
N
CH3
Lobelane
OH
OH
O
O
GZ-793A
Figure 1. Chemical structure of lobeline, lobelane, nor-lobelane, GZ-793A. Lobeline
is the principal alkaloid of Lobelia inflata. Lobelane is a chemically defunctionalized,
saturated derivative of lobeline. GZ-793A is a 4-methoxy analogue of nor-lobelane
incorporating an N-(2S)-1,2-dihydroxypropyl substituent.
The synthesis of the lobelane and GZ-793A derivatives utilized
compound 3 as a starting point (Scheme 1). Compound 3 was syn-
thesized by the reaction of 4-hydroxybenzaldehyde (1) with 2,
6-lutidine (2) in acetic anhydride at reflux temperature. Intermedi-
ately, 3 was subjected to hydrogenation using Adams catalyst
(PtO2) in acetic acid to yield a saturated piperidino derivative,
which was further converted to hydrochloride salt 4 using HCl in
diethyl ether. Compound 4 was reacted with NaBH3CN/HCHO to
obtain N-methylated compound 5. Compounds 4 and 5 were
de-acetylated in presence of K2CO3 in MeOH and followed by
hydrochloride salt formation to afford the corresponding free
hydroxyl compounds 6 and 7, respectively. Compound 4 was
reacted with S-glycidol in ethanol, followed by de-acetylation in
presence of K2CO3 in MeOH and formation of hydrochloride salt
to obtain the N-(2S)-1,2-dihydroxylpropyl derivative 8 (Scheme 1).
The fluorine-containing derivatives of lobelane and GZ-793A
were synthesized from intermediate 3. Compound 3 was de-acety-
lated in the presence of K2CO3 in MeOH to afford 9, which was
O-alkylated utilizing ethylfluorotosylate and sodium hydroxide
in DCM/MeOH (2:1) to yield a mixture of monofluoroethoxy
which exhibit such neurochemical profiles have the potential to
generate untoward side-effects in the clinical population. To over-
come these limitations, several structure–activity relationship
(SAR) studies were performed which involved modifying the
chemical structure of lobeline, and assessing the influence of these
structural alterations on selectivity of the novel compounds for
VMAT2 versus nicotinic receptors and plasmalemma neurotrans-
mitter transporters, DAT and the serotonin transporter (SERT).10
Lobelane is a structurally modified derivative of lobeline (Fig. 1),
which exhibits 10- to 15-fold higher potency for inhibiting VMAT2
function compared to lobeline.11 Importantly, structural defunc-
tionalization of lobeline to afford lobelane markedly diminished
nicotinic receptor affinity of lobelane and associated analogues.
Lobelane also inhibits METH-evoked DA release from rat striatal
slice preparations.11 To improve upon the physicochemical proper-
ties of lobelane, we synthesized a lobelane analogue, GZ-793A, by
structural modification of the N-methyl group in the molecule to
an N-(2S)-1,2-dihydroxyl-propyl moiety (Fig. 1). This structural
CHO
a
N
+
3
N
AcO
OAc
b, f
OH
2
1
N
c,f
HCl
N
H HCl
H
AcO
4
OAc
HO
OH
7
d, f
e, c, f
N
HCl
N
HCl
CH3
OH
OH
HO
OH
AcO
OAc
8
5
c, f
N
HCl
CH3
HO
OH
6
Scheme 1. Reagents and conditions: (a) Ac2O, reflux, 24 h, 48%; (b) 10% (w/v) PtO2/H2, AcOH, 12 h, 65%; (c) K2CO3/MeOH, rt, 1 h, 90–95%; (d) HCHO/NaBH3CN, EtOH, 2 h, 61%;
(e) (S)-glycidol, EtOH, reflux, 2 h, 57–60%; (f) 2 M HCl in ether.

2424
S. R. Joolakanti et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2422–2427
a
N
9
N
3
HO
OH
AcO
OAc
b
c, d
N
H
N
HCl
R2
R1
R2
R1
10 R1 = OH, R2 = OCH2-CH2-F
11 R1, R2 = OCH2-CH2-F
12
R
1 = OH, R2 = OCH2-CH2-F
13 R1, R2 = OCH2-CH2-F
f,d
e,d
N
N
HCl
CH3
HCl
OH
OH
R2
R1
R2
R1
14 R1 = OH, R2 = OCH2-CH2-F
15 R1, R2 = OCH2-CH2-F
1 = OH, R2 = OCH2-CH2-F
16
17
R
R , R2 = OCH2-CH2-F
1
Scheme 2. Reagents and conditions: (a) K2CO3/MeOH, rt, 1 h; (b) F-CH2-CH2-OTs, NaOH, DCM/MeOH (2:1), 2 h, 30–33%; (c) 10% (w/v) PtO2/H2, AcOH, 12 h, 60–65%; (d) DCM/
2 M HCl in ether; (e) HCHO/NaBH3CN, EtOH, 2 h 58–61%; (f) (S)-glycidol, EtOH, reflux, 2 h, 57–60%.
derivative 10 and difluoroethoxy derivative 11 (Scheme 2). After
careful separation of these two compounds by silica gel column
chromatography, 10 and 11 were each hydrogenated using Adams
catalyst (PtO2) in acetic acid to yield the corresponding saturated
piperidino derivatives, which were further converted to their
hydrochloride salts to obtain 12 and 13, respectively. Compounds
12 and 13 were then treated with NaBH3CN/HCHO to obtain the
N-methylated derivatives 14 and 15. N-(2S)-1,2-Dihydroxypropyl
derivatives 16 and 17 were obtained by reacting 12 and 13 with
S-glycidol in ethanol (Scheme 2). The synthesized compounds were
fully characterized by 1H NMR, 13C NMR and high resolution mass
spectral analysis.
Compounds 4–8 and 12–17 were evaluated initially for inhibi-
tion of VMAT2 function (Tables 1 and 2; Fig. 2). The majority of
Table 1
Inhibition of [3H]DA uptake at VMAT2 and DAT, and inhibition of [3H]5-HT uptake at SERT by precursor non-fluorinated lobelane analogues
Compound
VMAT2 [3H]DA Uptake (Ki;
lM)
DAT [3H]DA Uptake (Ki;
l
M)
SERT [3H]5-HT Uptake (Ki;
lM)
Lobelane
GZ-793A
0.067 0.007b
0.029 0.008b
1.05 0.03b
1.44 0.27b
3.6 0.35b
9.36 2.74b
N
H
0.22 0.021a
1.47 0.403
2.13 0.466
HCl
AcO
AcO
HO
OAc
OAc
4
N
0.071 0.008
2.13 0.87
11.6 1.46
HCl
CH3
5
N
HCl
CH3
0.046 0.005
0.031 0.004
1.35 0.34
12.0 2.00
1.03 0.17
OH
6
N
0.866 0.14
H HCl
HO
HO
OH
OH
7
N
HCl
OH
OH
0.039 0.003
1.43 0.73
12.9 2.98
8
a
Data represent mean SEM (n = 3–4 rats/analogue).
Data taken from Ref. 15 (Horton et al., 2011).
b

S. R. Joolakanti et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2422–2427
2425
M)
Table 2
Inhibition of [3H]DA uptake at VMAT2 and DAT, and inhibition of [3H]5-HT uptake at SERT by fluorinated lobelane analogues
Compound
VMAT2 [3H]DA Uptake (Ki;
lM)
DAT [3H]DA Uptake (Ki;
lM)
SERT [3H]5-HT Uptake (Ki;
l
Lobelane
GZ-793A
0.067 0.007b
0.029 0.008b
1.05 0.03b
1.44 0.27b
3.6 0.35b
9.36 2.74b
N
0.032 0.003a
0.040 0.002
0.031 0.003
0.14 0.011
1.66 0.34
1.67 0.30
2.97 0.46
3.43 0.73
1.05 0.29
0.640 0.034
10.4 0.54
H HCl
F
O
OH
12
N
H
HCl
F
F
O
O
13
N
HCl
F
CH3
O
OH
14
N
HCl
7.43 1.84
F
CH3
F
O
O
15
N
HCl
F
OH
OH
0.063 0.003
5.47 0.92
1.43 0.23
O
OH
16
N
HCl
F
OH
OH
F
0.120 0.003
3.69 0.72
33.0 2.92
O
O
17
a
Data represent mean SEM (n = 3–4 rats/analogue).
Data taken from Ref. 15 (Horton et al., 2011).
b
the compounds exhibited affinities for VMAT2 within a relatively
restricted range of 30–70 nM. Incorporation of 4-acetoxy sub-
stituents into the phenyl rings of nor-lobelane afforded 4, which
exhibited a Ki value of 0.22
of 4 afforded 5, which had threefold improved inhibitory potency
at VMAT2 (Ki = 0.071 M). Hydrolysis of the 4-acetoxy groups in
lM in the VMAT2 assay. N-methylation
l
4 and 5 gave the diphenolic analogues 7 and 6, respectively,
with about twofold further improvement in inhibitory potency
at VMAT2 (Ki = 0.031 lM and 0.046, respectively). These data indi-
cate that introduction of aromatic hydroxyl substituents into the
lobelane molecule retains the high inhibitory potency at VMAT2
exhibited by lobelane. Interestingly, this trend was also observed
for compound 8,
exhibited Ki of 0.039
a
diphenolic analogue of GZ-793A, which
M in the VMAT2 assay (Table 1).
l
Evaluation of nor-lobelane analogues 12 and 13, the mono-4-
(2-fluoroethoxy) and di-4-(2-fluoroethoxy) derivatives of 7, indi-
cated that replacing the phenolic groups in these compounds with
2-fluoroethoxy groups had little effect on inhibition of DA uptake
at VMAT2 (Ki’s = 0.032 and 0.040, respectively) in relation to
analogue 7. N-methylation of 12 afforded compound 14, one of
Figure 2. Lobelane and GZ-793A analogues inhibit [3H]DA uptake at VMAT2 in rat
striatal vesicles. Concentration–response curves for inhibition of VMAT2. Order of
presentation for the analogues within the figure legend is from high to low affinity
for VMAT2. Control (CON) represents [3H]DA uptake in the absence of compound.
Data are mean ( SEM) specific [3H]DA uptake expressed as a percentage of control
(24.8 1.06 pmol/min/mg, control n = 42 rats; n = 3–4 rats/compound).
the most potent inhibitors (Ki = 0.031
in the series. However, while the di-4-(2-fluoroethoxy) analogue
13 afforded a Ki value of 0.040 M, which is not different from that
of lobelane or GZ-793A, insertion of either a N-methyl group
lM) of vesicular DA uptake
l

2426
S. R. Joolakanti et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2422–2427
Figure 4. Lobelane and GZ-793A analogues inhibit [3H]5-HT uptake at SERT in rat
striatal synaptosomes. Concentration–response curves for inhibition of SERT. Order
of presentation for the analogues within the figure legend is from high to low
affinity for SERT. Control (CON) represents [3H]5-HT uptake in the absence of
Figure 3. Lobelane and GZ-793A analogues inhibit [3H]DA uptake at DAT in rat
striatal synaptosomes. Concentration–response curves for inhibition of DAT. Order
of presentation for the analogues within the figure legend is from high to low
affinity for DAT. Control (CON) represents [3H]DA uptake in the absence of
compound. Data are mean ( SEM) specific [3H]DA uptake expressed as a percentage
of control (19.5 1.27 pmol/min/mg, control n = 43 rats; n = 3–4 rats/compound).
compound. Data are mean ( SEM) specific [3H]5-HT uptake expressed as
a
percentage of control (4.46 0.234 pmol/min/mg, control n = 44 rats; n = 4
rats/compound).
(compound 15, Ki = 0.14
group (compound 17, Ki = 0.120
hydrogen atom in 13 reduces potency at VMAT2 by 3–3.5-fold. It
is important to note that compound 16, a mono-4-(2-fluoroethoxy)
analogue of GZ-793A, exhibited a Ki value of 0.063 lM in the
VMAT2 assay, and represents a lead fluorine-containing analogue
with improved water solubility over lobelane.
Potency of VMAT2 inhibition alone is insufficient to determine
whether the discovery of these novel lobelane analogues have clin-
ical potential as treatments for METH abuse. Accordingly, we also
examined the ability of these compounds to inhibit the plas-
malemma transporters, DAT and SERT (Tables 1 and 2; Figs. 3
and 4). Generally, all of the analogues in this study exhibited affini-
l
M) or
a
N-(2S)-1,2-dihydroxypropyl
In summary, a series of lobelane and GZ-793A analogues that
incorporate aromatic 4-hydroxy and aromatic 4-(2-fluoroethoxy)
substituents were synthesized and evaluated for their ability to
inhibit [3H]DA uptake at VMAT2 and DAT, and [3H]5-HT uptake
at SERT.16 Incorporation of these moieties into the structure of
lobelane generally maintained inhibitory potency at VMAT2 rela-
tive to the parent compound. The potent VMAT2 inhibitor 14,
which incorporates 4-(2-fluoroethoxy) and 4-hydroxy aromatic
substituents, exhibited the greatest selectivity for VMAT2 versus
DAT and SERT. These results also indicate that fluorinated lobelane
and GZ-793A analogues retain the high inhibitory potency and
selectivity of lobelane and GZ-793A for VMAT2, and as such consti-
tute promising candidates for the visualization of VMAT2 via PET
scanning in the clinical population.
l
M) in place of the piperidino
ties for DAT comparable to those for both lobelane (Ki = 1.05
and GZ-793A (Ki = 1.44
M).15 Likewise, a preponderance of the
compounds inhibited SERT with affinities not different from those
for lobelane (Ki = 3.6 M) and GZ-793A (Ki = 9.36
M).15 Notable
lM)
l
Acknowledgements
l
l
exceptions to these generalizations are compound 13 which has
sixfold greater affinity for SERT than lobelane, compound 16 which
has a sevenfold greater affinity for SERT than GZ-793A, and com-
pound 17 which exhibits a fourfold decrease in affinity for SERT
when compared to GZ-793A. Thus, compound 17 exhibits 30-
and 275-fold greater selectivity for VMAT2 versus DAT and SERT,
respectively. While incorporation of 2-fluoroethoxy moieties into
the structural scaffold of GZ-793A does not alter affinity for VMAT2
in comparison to both lobelane (Ki = 67 nM) and GZ-793A
(Ki = 29 nM), the greater selectivity for VMAT2 exhibited by these
compounds suggests a low likelihood of abuse liability.15
This research was supported by NIH U01 DA13519, 5T32
DA022981, TR000117 and NIH/COBRA P20 GM109005 grants,
and an Arkansas Research Alliance (ARA) Scholar award.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03.
119.
References and notes
Similar to lobelane and GZ-793A, all the compounds evaluated
in this study exhibited higher affinities for VMAT2 than DAT and
SERT. Most compounds showed 15 to 40-fold greater selectivity
for VMAT2 versus the plasmalemma transporters. The combination
of 4-(2-fluoroethoxy) and 4-hydroxy aromatic substitution present
in compounds 14 and 16 afforded the greatest selectivity for
VMAT2 versus DAT, 96- and 87-fold respectively. Lobeline
analogue 14 was one of the most potent VMAT inhibitors in the
series. Likewise, incorporation of 4-acetoxy and 4-hydroxy aro-
matic substituents into the phenyl rings of lobelane resulted in
improvement in selectivity for VMAT2 versus SERT; compounds
5, 6 and 8 exhibited selectivity ratios of 163, 260- and 331-fold,
respectively. Overall, compound 14 demonstrated 96- and 335-fold
selectivity for VMAT2 versus DAT and SERT, respectively, and was
the most selective analogue of the series.
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terminated by filtration, and radioactivity retained by the filters was
determined by liquid scintillation spectrometry (Liquid scintillation analyzer;
PerkinElmer Life and Analytical Sciences, Boston, MA).
Synaptosomal [3H]DA and [3H]5-HT Uptake Assays. Analogue-induced inhibition
of [3H]DA and [3H]5-HT uptake into rat striatal synaptosomes, was determined
using modifications of a previously described method.15 Striata were homog-
enized in 20 ml of ice-cold 0.32 M sucrose solution containing 5 mM NaHCO3,
pH 7.4, with 16 up-and-down strokes of
a Teflon pestle homogenizer
(clearance ꢀ0.005 inch). Homogenates were centrifuged at 2000g for 10 min
at 4 °C, and resulting supernatants were centrifuged at 20,000g for 17 min at
4 °C. Pellets were re-suspended in 1.5 mL of Krebs’ buffer, containing 125 mM
NaCl, 5 mM KCl, 1.5 mM MgSO4, 1.25 mM CaCl2, 1.5 mM KH2PO4, 10 mM a-D-
glucose, 25 mM HEPES, and 0.1 mM EDTA, with 0.1 mM pargyline and 0.1 mM
ascorbic acid, saturated with 95% O2/5% CO2, pH 7.4. Synaptosomal suspen-
13. (a) Alvers, K. M.; Beckmann, J. S.; Zheng, G.; Crooks, P. A.; Dwoskin, L. P.; Bardo,
M. T. Psychopharmacology (Berl.) 2012, 224, 255; (b) Beckmann, J. S.; Denehy, E.
D.; Zheng, G.; Crooks, P. A.; Dwoskin, L. P.; Bardo, M. T. Psychopharmacology
(Berl.) 2012, 220, 395.
14. Elliott, A. J.; Hudlicky, M.; Pavlath, A. E. ACS Monograph 1995, 187, 1119.
15. Horton, D. B.; Siripurapu, K. B.; Zheng, G.; Crooks, P. A.; Dwoskin, L. P. J.
Pharmacol. Exp. Ther. 2011, 339, 286.
sions (20
lg of protein/50 lL) were added to duplicate tubes containing 50 lL
of analogue (7–9 concentrations; 0.1 nM–1 mM, final concentration) and
350
450
l
l
L
of buffer and incubated at 34 °C for 5 min in
L. Samples were placed on ice, and 50
L of [3H]DA or [3H]5-HT (10 nM,
L.
a total volume of
16. Vesicular [3H]DA Uptake Assay. Inhibition of [3H]DA uptake was conducted as
previously described,8b utilizing a striatal synaptic vesicle preparation. Briefly,
rat striata were homogenized with 10 up and down strokes of a Teflon pestle
homogenizer (clearance ꢀ0.00300) in 14 mL of 0.32 M sucrose solution.
Homogenates were centrifuged (2000g for 10 min at 4 °C), and the resulting
supernatants were centrifuged again (10,000g for 30 min at 4 °C). Pellets were
resuspended in 2 ml of 0.32 M sucrose solution and subjected to osmotic shock
by adding 7 mL of ice-cold water to the preparation, followed by the
l
final concentration) was added to each tube for a final volume of 500
l
Reactions proceeded for 10 min at 34 °C and were terminated by the addition
of 3 mL of ice-cold Krebs’ buffer. Nonspecific [3H]DA and [3H]5-HT uptake were
determined in the presence of 10 lM GBR 12909 and 10 lM fluoxetine,
respectively. Samples were rapidly filtered through Whatman (Clifton, NJ) GF/B
filters using a cell harvester (MP-43RS; Brandel Inc., Gaithersburg, MD). Filters
were washed three times with 4 mL of ice-cold Krebs’ buffer containing
catechol (1 mM). Complete counting cocktail was added to the filters, and
radioactivity was determined by liquid scintillation spectrometry (B1600 TR
scintillation counter; PerkinElmer Life and Analytical Sciences
immediate restoration of osmolality by adding 900
buffer and 900 of 1.0 M potassium tartrate solution. Samples were
centrifuged (20,000g for 20 min at 4 °C), and the resulting supernatants were
lL of 0.25 M HEPES
lL