Figure 9.
Overnight incubation with high-concentration opioids decreased acute Gαi activation and βarr2 recruitment. Transfected HEK293T cells were treated overnight with vehicle, high-concentration (10 μm) DADLE or DAMGO, washed for 30 min, and then stimulated with vehicle, or agonist with low-concentration (10 nm) DAMGO or DADLE or high-concentration (10 μm) DAMGO or DADLE. BRET was recorded over time to measure activation of Gαi (A: F(5,24) = 45.25, p < 0.0001; B: F(5,24) = 99.2, p < 0.0001; C: F(5,23) = 0.1771, p = 0.9685; D: F(5,23) = 51.16, p < 0.0001), Gαs (E: F(5,24) = 0.8942, p = 0.5007; F: F(5,24) = 0.1545, p = 0.9766; G: F(5,23) = 0.9256, p = 0.4824; H: F(5,23) = 0.477, p = 0.7896), and Gαq (I: F(5,24) = 7.611, p < 0.0002; J: F(5,24) = 8.63, p < 0.0001; K: F(5,23) = 1.63, p = 0.1919; L: F(5,23) = 6.034, p = 0.001) by dissociation of Rluc2-Gα with GFP10-Gγ2 after agonist stimulation. Consequently, a decrease of BRET reflects an activation of Gα protein. Following the same cell treatment, recruitment of Rluc2-βarr2 to the plasma membrane (rGFP-CAAX) was also measured using ebBRET (M: F(5,24) = 8.966, p < 0.0001; N: F(5,24) = 24.1, p < 0.0001; O: F(5,23) = 0.7804, p = 0.574; P: F(5,23) = 13.28, p < 0.0001), an increase of ebBRET reflecting the recruitment of βarr2 at the plasma membrane. Data were collected from 3-7 independent experiments. Bars represent mean of the area under the curve ± SEM. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.001; one-way ANOVA with multiple comparisons and Tukey's post hoc test.