Fig 5.
Relationship of receptor occupancy to response. (A) Affinity of epinephrine (Epi, ○), moxonidine (Mox, ▾), dexmedetomidine (Dex, ▵), and clonidine (Clon, ▪) for the α2A-AR was assessed by using competition for [3H] Yohimbine binding under conditions where alterations in receptor affinity caused by receptor coupling to G proteins would not occur (see Materials and Methods). Data are presented as % occupancy (calculated as 100% - % competition for [3H] Yohimbine binding), and are the mean ± SEM for 5–10 independent experiments. (B) Agonist-stimulated GTP[γ35S] binding to G proteins in membrane preparations derived from cells expressing HA-α2A-AR at 7.6 pmol α2A-AR/mg protein is presented as % of control for epinephrine (○), moxonidine (▾), dexmedetomidine (▵), and clonidine (▪), where control represents GTP[γ35S] binding in the absence of agonist. Shown in parentheses in B–D for each curve are % response relative to that of epinephrine (defined as 100%). Data represent the mean ± SEM for 6–12 separate experiments. (C and D) Agonist-stimulated MAP kinase activity in cells expressing HA-α2A-AR at 0.82 pmol α2A-AR/mg protein (C) and at 7.6 pmol α2A-AR/mg protein (D) was calculated as [(p42 and p44 ACTIVE MAP kinase) ÷ (p42 and p44 TOTAL MAP kinase) − baseline (no agonist)] for epinephrine (○), moxonidine (▾), dexmedetomidine (▵), and clonidine (□), and was normalized to % of maximal epinephrine stimulation (maximal epinephrine stimulation: 22.9 arbitrary units = 100% in C, 48.0 arbitrary units = 100% in D). The data shown are mean ± SEM for 3 (C) and 4 (D) independent experiments performed under identical conditions. (E and F) Representative Western blots for ACTIVE (Upper) and TOTAL (Lower) MAP kinase activity. No stimulation of MAP kinase by the agonists evaluated is observed in parental cells serving as the background for the HA-α2A-AR-expressing cells. E and F correspond to a representative experiment performed in cells expressing the α2A-AR at 0.82 pmol/mg protein and 7.6 pmol/mg protein, respectively.