Fig. 1.

μ and δ receptor interactions in heterologous cells and endogenous tissue. (A) BRET assay shows significant energy transfer between μ and δ receptors in live cells. (Upper) Light emission was monitored for cells transfected with δLuc and CCR5YFP (long black dash) or various amounts of δLuc:μYFP; 0.1:0.8 (gray line), 0.25:2 (gray dash), and 0.5:4 (small black dash). Spectra in A expressed as BRET ratio, defined as the ratio of the area under the curve of light emission for δLuc:μYFP and the curve of light emission for δLuc alone, is shown in the Inset. (Lower) Light emission was monitored for cells coexpressing μLuc with either CCR5YFP (dash), or δYFP (black), or YFP (gray). Spectra in B expressed as BRET ratio is shown in Inset. (B) μ and δ receptor complexes can be isolated from spinal cord membranes. Solubilized spinal cord membranes from WT mice or mice lacking δ receptors (δ k/o) were subjected to immunoprecipitation by using 1 μg of anti-μ or anti-δ mAbs (μ-mAb or δ-mAb). Western blotting of immunocomplexes isolated by using anti-μ or anti-δ mAbs with δ polyclonal antibodies (Chemicon) detected δ receptors in membranes from WT mice but not from mice lacking δ receptors. Western blotting of immunocomplexes isolated by using anti-δ mAbs antibodies with μ polyclonal antibodies (gift from T. Cote) detected μ receptors in membranes from WT mice but not from mice lacking δ receptors (data not shown). (C and D) δ antagonists modulate the binding of ³H-DAMGO and ³H-morphine. (C) CHO cells stably expressing μ receptors or coexpressing μ and δ opioid receptors were incubated with increasing doses of ³H-DAMGO in the absence or presence of various ligands (10 nM). The incubations were carried out for 2 h at 37°C. Cells were washed in ice-cold 50 mM Tris·HCl, and radioactivity bound was detected as described (13). Results are mean ± SEM of three experiments in triplicate. (D) CHO cells stably expressing μ receptors, coexpressing μ and δ opioid receptors or SK-N-SH cells endogenously expressing these receptors were incubated with increasing doses of ³h-damgo or ³H-morphine in the absence or presence of various ligands (10 nM), and the radioactivity bound was detected as described (13). Results are mean ± SEM of three experiments in triplicate. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001, Dunnett's test. NA, not available.