Figure 4

WIN55,212-2 promotes reversible transfer of CB1R-regulated G proteins toward RGSZ2 proteins. A. Gα subunits co-precipitated with CB1Rs in PAG membranes. These proteins were assayed using antibodies to different Gα subunits. Gαz was studied using an antibody targeting the helical domain (1) and an antibody targeting the C-terminus (2). B. Groups of mice received a single icv injection of WIN55,212-2 (20 nmol) and were killed at the time points indicated. For each time point studied the PAG structures from 6 to 8 mice were pooled. The CB1R, RGSZ2, and associated proteins were then studied in PAG membranes. The immunosignals associated with these signals (average optical density of the pixels within the object area/mm2; Quantity One Software, BioRad) were expressed relative to the levels observed for the control group (attributed an arbitrary value of 1). Each bar is the mean ± SEM of three assays performed on PAG samples obtained from independent groups of mice. *Significantly different from the PAG control group (P < 0.05). C. Stimulated [³⁵S]GTPγS binding by WIN55,212-2 in PAG membranes from mice injected with a cannabinoid agonist. Membranes from mice treated with vehicle (control; closed symbols) or exposed to WIN55,212-2 (20 nmol/mouse; open symbols) were obtained 30 min and 48 h after icv injection and incubated in [³⁵S]GTPγ S (0.1 nM) and increasing concentrations of WIN55,212-2. *Significantly different from the PAG control group (P < 0.05).