Desensitization of cAMP inhibition and superactivation of AC after pretreatment with spiradoline (A) Spiradoline (1 nM pretreatment) inhibited forskolin-induced cAMP formation in HEK293 cells transiently expressing Rog, Rog-A, Rog-μ, and Rog-μA. Data are expressed as inhibition of forskolin-induced cAMP. The baseline (0) represents maximum forskolin-induced cAMP accumulation in control cells. Pretreatment with 1 nM spiradoline for 10 min causes a shift in the dose-response curve for later spiradoline treatment in Rog and Rog-μA cells, but not Rog-A. Spiradoline pretreatment lowered the maximal response of Rog-μ to subsequent spiradoline treatment. (B) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for cAMP accumulation in response to a 15-min treatment with 10 μM forskolin. Spiradoline pretreatment significantly increased forskolin-induced cAMP in cells expressing KOR, Rog and Rog-A. Pretreatment of cells expressing Rog-μ and Rog-μA had not effect on response to forskolin. Data are expressed relative to the amount of cAMP accumulated after 10 μM forskolin treatment in cells pretreated with vehicle. Bars represent mean ± SEM for six replicates per condition. (C) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for loss of cell-surface expression by ELISA. Long-term spiradoline treatment significantly reduces cell-surface expression of all receptors, but the degree of internalization does not differ among different receptors. Bars represent mean ± SEM for three replicates per condition.