In the present study we have used [³H]-SR141716A (Rinaldi-Carmona et al., 1994) and [³⁵S]-GTP--S binding to study cannabinoid CB₁ receptors expressed in recombinant HEK293 cells and in rat cerebellar membranes.

Rat (Sprague Dawley) cerebellum or HEK293 cell membranes expressing human CB₁ receptors were suspended in 50 mM Tris HCl (pH 7.6, 5 mM MgCl₂, 2 mM EDTA, 0.5mg/ml BSA). Assays were incubated at 30°C for 90 min in 250 µl containing 40 µg of protein and 1nM [³H]-SR141716A. Non-specific binding was defined with 100 µM CP55940. For GTP--S binding rat cerebellar membranes were suspended in 20 mM HEPES, (3 mM MgCl₂, 60 mM NaCl₂, 1 mM EGTA, 0.5mg/ml BSA, and various concentrations of GDP) and tissue was incubated with 0.2 nM [³⁵S]-GTP--S, as above. [³H]-SR141716A bound to receptors in rat cerebellum (Kd: 0.61 ± 0.13 nM, Bmax: 1.43 ± 0.64 pmol/mg protein, mean ± SEM, n= 3) and to HEK-293 cells (Kd: 0.82 ± 0.17 nM, Bmax: 13.6 ± 2.37 pmol/mg protein). Ki's for displacers are shown in Table 1.
Table 1. Ki values for compounds displacing [³H]-SR141716A from rat cerebellar membranes and HEK 293 cells expressing human recombinant CB₁ receptors. Data is expressed in nM (mean ± SEM, n=3 independent experiments).

Ligand	Rat cerebellum	HEK-293
AM251 (Gatley et al., 1997)	1.17±0.26	0.72±0.04
SR141716A (Rinaldi-Carmona et al., 1994)	2.25 ± 0.41	2.38±0.33
WIN55212-2 (D'Ambra et al.,1992)	161± 22	412±37
CP55940 (Herkenham et al., 1990)	54.4 ± 20	20.6±3.4
9-tetrahydrocannabinol	2460 ± 753	257 ± 30

Eight concentrations of GDP were used to determine conditions for [³⁵S]-GTP--S binding. Basal [³⁵S]-GTP--S binding at 1 µM GDP was 3917±115 cpm (mean ± SEM, n=3); this was reduced to 712±35 cpm in the presence of 200 µM GDP. SR141716A inhibited basal [³⁵S]-GTP--S binding. The pEC₅₀ for this response was increased from 7.52 ± 0.22 (mean ± SEM, n= 3) at 1µM GDP to 8.69 ± 0.4 at 50 µM GDP (p<0.05, Student's t-test). If SR141716A acts as an antagonist of an endogenous CB₁ agonist, an increase in the potency of the endogenous agonist would explain this shift. Such a shift in potency is likely to be reflected in a decrease in the pEC₅₀ of exogenously applied agonist. This shift was not observed: the pEC₅₀ for CP55940 was 8.03 ± 0.36 and 8.00 ± 0.49 (1mM and 50 mM GDP, respectively); the pEC₅₀ for WIN55212-2 was 7.03 ± 0.26 and 7.01 ± 0.22. That GDP modulated the activity of SR141716A without changing agonist EC₅₀ may indicate that SR141716A acts as an inverse agonist.

D'Ambra T E. et al. (1992). J. Med. Chem. 35(1), 124-35.
Gatley S J. et al. (1997).Life Sciences 61(14), 191-7.
Herkenham M. et al. (1990). Proc. Natl. Acad. Sci. 87(5), 1932-6.
Rinaldi-Carmona M. et al. (1994). FEBS Letts 350 (2-3), 240-4.