Cannabinoid CB1 Receptor Radioligand Binding

Cannabinoid receptor ligands are generally highly lipophilic, and the concentrations that can be used are limited by this fact (see also Notes 2 and 3). A sample saturation assay of the commonly used CB1-selective antagonist SR141716A is illustrated in Fig. 1. The figure shows how concentrations of this ligand above 5 nM result in more nonspecific binding than specific binding, and thus rarely yield usable results. This is true even in rat cortex membranes where CB1 is expressed at very high levels (compared to other GPCRs in brain); in this assay, the Bmax of [3H]SR141716A binding was around 5 pmol/mg. The ratio of specific to nonspecific binding can be improved by increasing the amount of membrane protein in the assay, but doing so also increases the chances of lig-and depletion; in fact, in the assay shown in Fig. 1, more than 10% of total lig-and was bound at the four lowest concentrations. Decreasing the amount of protein to avoid this complication will limit the useful maximum concentration of ligand further.

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Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [3H]SR141716A (a CB1-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nM) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 |g/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 |M unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [3H]SR141716A.

[3H]SR141716A added (nM)

Fig. 1. Saturation binding of a cannabinoid receptor ligand to brain membranes. Shown are the results of a typical saturation assay of [3H]SR141716A (a CB1-selective antagonist) in rat cortex membranes. Various concentrations (0.05-5.5 nM) of radioligand were incubated in assay buffer with 0.1% BSA and rat cortex membrane homogenates (20 |g/tube) for 1 h at 30°C in the absence (total DPM) and presence (nonspecific DPM) of 5 |M unlabeled SR141716A. Specific DPM and nonspecific DPM shown are the mean of three values measured in consecutive assay tubes in the same rack. Specific DPM were calculated by subtracting mean nonspecific DPM from mean total DPM at each concentration of [3H]SR141716A.

3.2.1. Receptor Binding Procedure

1. Tris buffer (50 mM Tris-HCl, pH 7.4) with 0.2% (w/v) BSA is added to test tubes on ice. To duplicate the conditions of the [35S]GTPyS binding assay, assay buffer (50 mM Tris, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 100 mM NaCl) with the same concentration of GDP and GTPyS as used in the [35S]GTPyS-binding assay could be used with antagonist radioligands. This does not work very well with agonist radioligands, since NaCl and GTPyS decrease specific binding of GPCR agonists.

2. Unlabeled cannabinoid ligands (for nonspecific binding) are added to the assay tubes. Nonspecific binding is determined in the presence of unlabeled ligand approx 1000-fold higher than that of the labeled ligand (usually 1 |M; see Note 2).

3. Membrane homogenates (prepared as above) from CNS tissues (10-50 |g/tube) or cultured cells expressing cannabinoid receptors (5-100 |g/tube, depending on the level of expression) are added to the assay tubes.

4. Add approx 0.01-5 nM cannabinoid receptor radioligand (see Notes 2 and 3) for a final volume of 1.0 mL.

5. Initiate the assay by incubating (typically in a temperature-controlled, shaking water bath) for 1-2 h at 30°C, depending on the ligand.

6. Terminate by rapid filtration onto Whatmann GF/B glass fiber filters that have been soaked for at least 1 h in 50mM Tris-HCl, pH 7.4, containing 0.5% BSA. Filters are then rinsed seven times with approx 3 mL/tube Tris-HCl, pH 7.4, containing 0.05% BSA.

7. Place filters in scintillation vials with several milliliters of scintillation fluid, and allow the samples to extract into the scintillation fluid overnight (or shake for 1 h).

8. Determine radioactivity by liquid scintillation spectrophotometry.

Each condition in this assay is repeated in triplicate. Mean values ± SD are calculated for each triplicate, and triplicates for which the SD is greater than 10% of the mean are inspected for values that are outliers.

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