Figure 4.

Immunocomplexes containing μ receptors (μOR) and D₁ receptors (D1R) and functional interaction of two receptors were present in rodent striatum. (A) Heterodimeric complexes of μ receptors and D₁ receptors were found in mouse striatal membranes. Solubilized striatal membranes from mice were subjected to immunoprecipitation (IP), using anti‐μ receptor or anti‐D₁ receptor antibodies, as described. Immunocomplexes isolated by using anti‐μ receptor or anti‐D₁ receptorantibody were immunoblotted (WB) by using anti‐D₁ receptor or anti‐μ receptor antibodies. A band of 130 kDa is seen only upon administration of anti‐D₁ receptor or anti‐μ receptor antibodies (lanes 2 and 4) and not in administration of non anti‐D₁ receptor or anti‐μ receptor antibody vehicle control (lanes 1 and 3). (B) Effect of SCH23390 on DAMGO‐stimulated [³⁵S]GTPγS binding to mouse striatal membranes prepared from wild‐type and D₁ receptor KO mice. Striatal membranes prepared from wild‐type mice or D₁ receptor KO mice were treated with 1 μM DAMGO with or without 1 μM SCH23390, and [³⁵S]GTPγS binding was measured as described. Values are expressed as the mean ± SEM of three independent experiments performed in triplicate. (C) SCH23390 suppressed DAMGO‐induced activation of ERK. Cultured striatal neurons were treated with DAMGO (1 μM) for 15 min in the absence or presence of SCH23390 (1 μM). The phosphorylation of ERK1/2 was determined by immunoblotting and quantified by densitometry as described. Top panel shows representative blots from Western blotting (WB); lower panel shows quantification of blots of phospho‐ERK1/2. Values are expressed as the mean ± SEM of four independent experiments. (D) SCH23390 failed to attenuate morphine‐induced activation of ERK1/2 in D₁ receptor KO mice. D₁ receptor KO mice were treated with morphine (10 mg·kg⁻¹, s.c.) with or without SCH23390 (0.03 mg·kg⁻¹, i.p.) for 2 h. SCH23390 was given 10 min prior to morphine administration. (E, F) SCH23390 attenuated morphine‐induced c‐Fos expression in wild‐type but not D₁ receptor KO mice. Wild‐type and D₁ receptor KO mice were treated with morphine (10 mg·kg⁻¹, s.c.) with or without SCH23390 (0.03 mg·kg⁻¹, i.p.) for 2 h. SCH23390 was given 10 min prior to morphine administration. Extracts of mouse striatum (50 μg) were subjected to SDS‐PAGE and immunoblotted by using rabbit anti‐c‐Fos (1:500). The intensities of the immunoreactive bands were quantified by densitometry. Top panel shows representative blots from Western blotting. Lower panel shows quantification of blots of c‐Fos. All values are normalized by using actin as a control protein. Values are expressed as the mean ± SEM (n = 6). *P < 0.05, significantly different from saline‐treated control group; ^# P < 0.05, significantly different from morphine‐treated group.