SUPPLEMENTARY MATERIALS
Materials and methods
Cell culture
HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented
with 4.5 mg/ml glucose and 0.11 mg/ml sodium pyruvate, 10% fetal bovine serum,
100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, (all supplements
were purchased from Life Technologies Europe BV, Stockholm, Sweden) at 37°C in
a humidified atmosphere of 5% CO2. Cells were passed when they were 80-90%
confluent. All cell culture reagents were purchased from Life Technologies Europe.
Cell transfection
HEK293 cells were grown to 70-80% confluence in 75cm2 tissue culture flasks and
transfected with the human -1 receptor (synthesized by GenScript Inc., Piscataway,
NJ; identical to GenBank accession number: NM_005866) in the pXOOM vector
using linear polyethylenimine, MW 25 000 (PEI, Polysciences Europe GmbH,
Eppelheim, Germany). Briefly, the plasmid DNA was diluted in culture medium
containing no additives (serum, antibiotics or other protein), and PEI was added (ratio
µg DNA: µg PEI, 1:7.5) and incubated for 8 min at room temperature. Culture
medium containing 10% FBS was added to the DNA/PEI complex, and the mixture
was applied to the cultures. After 4 h of incubation, the culture medium was replaced
with fresh medium. Transfected cell cultures were harvested 48 h post transfection by
washing twice with ice-cold PBS and subsequently scraped from tissue culture vessels
into the same buffer. The cells were centrifuged at 500  g for 10 min at 4°C and
resuspended in preparation buffer [PB; 50 mM Tris-HCl, pH 7.4, containing a
cocktail of protease inhibitors (Roche Diagnostics Scandinavia AB, Bromma,
Sweden) and centrifuged again. The cell pellets were sonicated in 2 ml PB in a ice-
bath to avoid heating of the samples and then centrifuged at 40,000  g for 60 min at
4°C. The pelleted membranes were resuspended and sonicated in the same PB and
centrifuged again. The protein concentration was determined for the pelleted
membranes by using the BCA Protein Assay (Pierce, Rockford, IL, USA) using
bovine serum albumin (BSA) dilutions to construct a standard curve. Pelleted
membranes were resuspended to a concentration of 2 mg/ml and aliquots were stored
at -80 °C.
Animals
All studies involving animals were performed in accordance with guidelines from the
Swedish National Board for Laboratory Animals. Male Sprague-Dawley rat (CharlesRiver, Germany) striatal membranes were prepared for their use in radioligand
binding experiments. Rats were sacrificed by decapitation and the brains were
removed and cooled for 2 min in ice-cold saline. The dorsal striatum from each
animal was dissected, removed and immediately frozen on dry ice and later stored at 80 °C until future use. Dorsal striatal tissue was placed in 10 ml of preparation buffer
[PB; 50 mM Tris-HCl, pH 7.4, containing NaCl (100 mM), MgCl2 (7 mM) and
EDTA (1mM)] containing a cocktail of protease inhibitors (Roche Diagnostics,
Mannheim, Germany). The tissue was homogenized using a basic IKA T18
mechanical tissue homogenizer (IKA Werke, Staufen, Germany) and centrifuged at
40,000  g for 40 min at 4 °C. The pelleted membranes were resuspended and
homogenized in the same PB and centrifuged an additional 3 times. The protein
concentration was determined for the pelleted membranes by using the BCA Protein
Assay (Pierce, Rockford, IL, USA) using bovine serum albumin (BSA) dilutions to
construct a standard curve. Pelleted membranes were resuspended to a concentration
of 2 mg/ml and aliquots were stored at -80 °C.
Radioligand binding experiments
[3H](+)-pentazocine binding assays
The -1 receptor agonist [3H](+)-pentazocine (6.0-9.2 nM; specific activity 34.8
Ci/mmol, Perkin-Elmer, Waltham, MA, USA) binding was displaced by 12 to 13
concentrations of either (+)-3-PPP (Sigma-Aldrich, St. Louis, MO, USA), ACR16
(synthesized by Axon MedChem BV, Groeningen, the Netherlands), (-)-OSU6162
(Tocris Bioscience, Bristol, United Kingdom), (-)-apomorphine (Sigma-Aldrich) or
bromocriptine (Sigma-Aldrich) to determine their affinities at -1 receptors from the
competition curves obtained. The Kd value of (+)-pentazocine for -1 receptors was
determined through radioligand saturation experiments using 8 concentrations (0.5 40 nM) of the -1 receptor agonist [3H](+)-pentazocine (34.8 Ci/mmol) and was
subsequently used to calculate the Ki values (presented in Table 1) from the
competition curves obtained. Experiments were performed using membrane
preparations from transfected HEK293 cells (80 μg protein/ml) or dorsal striatal
membranes from rat (240 μg protein/ml) and were incubated for 90 min at 25 °C in
incubation buffer [IB; 50 mM Tris-HCl pH 7.4]. The incubation was terminated by
rapid filtration through GF/C filters using a Brandel cell harvester with three washes
of 5 ml of ice-cold wash buffer (WB; 50 mM Tris-HCl pH 7.4). Nonspecific binding
was defined by radioligand binding in the presence of 10 μM haloperidol (SigmaAldrich). The radioactivity content of the filters was detected by liquid scintillation
spectrometry.
Data and statistical analysis
All radioligand binding data were analyzed using GraphPad PRISM 5.0 (GraphPad
Software Inc., La Jolla, CA) and where an alpha level of 0.05 was used for all
statistical tests. Nonspecific binding of [3H](+)-pentazocine to membrane preparations
in the presence of 10 μM haloperidol was subtracted from the total radioactivity to
determine specific [3H](+)-pentazocine binding. In competition binding experiments,
the specific binding was normalized to the amount of specific binding in the absence
of displacing ligand. Data was fitted to one- or two-binding site models as determined
by analyses of variance (F-tests). The significance of the fit with two- as compared to
one-site binding models, as well as whether the Hill slope of a one-site fit was
significantly different from -1, were tested. The inhibition constants (Ki, or KiH and
KiL) were calculated from IC50 values derived from the fits to competition binding
data as described by Cheng and Prusoff (1973).
Results
Membranes were prepared from HEK293 cells that were transfected with the human
-1 receptor and ligand binding characteristics were determined by both saturation
and competition assays using the specific -1 receptor agonist [3H](+)-pentazocine.
The Kd value of [3H](+)-pentazocine for recombinantly expressed human -1
receptors was determined to be 20.8 ± 2.6 nM with an expression level (Bmax) of 4.9 ±
0.3 pmol/mg protein. The dopamine stabilizers (-)-OSU6162 and ACR16 were
compared to the classical -1 receptor ligand (+)-3-PPP (see Fig. 1a for compound
structure). ACR16 displaced [3H](+)-pentazocine with similar affinity as a classical 1 receptor ligand, (+)-3-PPP, while (-)-OSU6162 was ~ 4 times less affine (Fig. 1b;
Table 1). Both (+)-PPP and ACR16 displacement of [3H](+)-pentazocine was
monophasic while (-)-OSU6162 displacement of [3H](+)-pentazocine was biphasic
(significantly fitting better than monophasic; F(2,70) = 6.589, p < 0.01).
Membrane fractions were also prepared from homogenized rat dorsal striata to
determine the affinity of ACR16 and (-)-OSU6162 at -1 receptor binding sites in rat
brain. The Kd of [3H](+)-pentazocine at endogenously expressed rat -1 receptors was
determined to be 10.4 nM ± 2.3 with a Bmax of 174.8 ± 15.8 fmol/mg protein. Both
ACR16 and (-)-OSU6162 displacement of [3H](+)-pentazocine was biphasic
(significantly fitting better than monophasic; F(2,184) = 8.606, p < 0.001 and F(2,76)
= 5.788, p < 0.01, respectively). High (KiH) and low (KiL) affinity constants of ACR16
and (-)-OSU6162 at -1 receptors in rat striatal membranes were calculated (Fig. 1c;
Table 1). In addition to the dopaminergic stabilizers, two clinically used agonists were
evaluated for their affinity at -1 receptors in rat striatal membranes. Both (-)apomorphine and bromocriptine demonstrate μM affinity for -1 receptors resulting
in a monophasic displacement of [3H](+)-pentazocine (Fig. 1c; Table 1).
References
Cheng Y, Prusoff WH. Biochem Pharmacol 1973; 22: 3099-3108.