Binding
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final
volume of 1 mL, with rodent brain preparations (0.3–0.5 mg
protein) or membranes from CHO cells expressing the human opioid receptors
(15–20 μg) and various concentrations of test compound
as described previously.44 (link),49 (link),64 (link) Rat brain membranes were incubated with either [3H]DAMGO
(1 nM, 45 min, 35 °C) or [3H][Ile5,6]deltorphin
II (0.5 nM, 45 min, 35 °C) for labeling MOR or DOR receptors,
respectively. Guinea-pig brain membranes were incubated with [3H]U69,593 (1 nM, 30 min, 30 °C) for labeling KOR. Binding
assays with CHO cell membranes were conducted at 25 °C for 60
min using [3H]DAMGO (1 nM) or [3H]diprenorphine
(0.2 nM) for labeling MOR or DOR, respectively. Nonspecific binding
was determined using 10 μM naloxone (rodent brain) or 1–10
μM of the unlabeled counterpart of each radioligand (CHO cells).
Reactions were terminated by rapid filtration through Whatman glass
fiber GF/C filters. Filters were washed three times with 5 mL of ice-cold
50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester
(Gaithersburg, MD). Radioactivity retained on the filters was counted
by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman
Coulter Inc., Fullerton, CA). All binding experiments were performed
in duplicate and repeated at least three times.
