FIGURE 5.

Co-activation with βAR subtype-selective agonists accelerates α₂AR agonist-mediated α_2AAR endocytosis in native neurons. A, neurons (SpWT) were subjected to treatment with a panel of clinically relevant agonists with varying degrees of β₁ versus β₂AR subtype selectivity (1 μm DOB, 1 μm ALB, or 100 nm SAL) and 10 min of stimulation with clonidine (1 μm). α_2AAR endocytosis (detected by prelabeling method), as indicated by the appearance of intracellular punctae containing internalized receptors (arrows), was observed with co-activation by DOB, ALB, and SAL, but not with clonidine alone. B, quantitation of agonist-mediated α_2AAR endocytosis, with relative internalization determined as described under “Experimental Procedures.” Internalization by clonidine alone was significantly enhanced with co-activation by DOB, ALB, and SAL. The data for clonidine plus ISO from Fig. 4 are included for comparison. Confocal images are representative of at least three independent samples, and quantitation was performed over at least 12–14 neurons (except for clonidine plus SAL, n = 10). *, p < 0.01 versus clonidine alone. †, p < 0.01 versus clonidine plus DOB or clonidine plus ALB.