β-Agonist-induced constitutive β2-adrenergic receptor activity in bovine tracheal smooth muscle

Abstract

1. According to the two state receptor model, the β2-adrenergic receptor (β2-AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G-protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term β2-AR activation by fenoterol on constitutive receptor activity. 2. Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 μM fenoterol, followed by extensive washout (3 h, 37°C), caused a rapid and time-dependent inhibition of KCl-induced contraction, reaching 68 ± 10, 51 ± 6 and 46 ± 4% of control, respectively, at 40 mM KCl (P < 0.05 all). At all time points, the EC50 values to KCl were significantly reduced as well. 3. Preincubation of BTSM with 0.1, 1.0 and 10 μM fenoterol during 18 h caused a concentration-dependent decrease of the 40mM KCl response to 70 ± 5, 47 ± 12 and 43 ± 9% of control, respectively (P < 0.05 all). 4. The reduced KCl contractions were reversed in the presence of 1 μM timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other β-blockers, with a rank order of efficacy of pindolol ≥ timolol = propranolol > alprenolol ≥ sotalol > labetalol. 5. At 25 mM KCl-induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol-induced effects cannot be explained by residual β-agonist binding. 6. In conclusion, fenoterol treatment of BTSM causes a time- and concentration-dependent development of constitutive β2-AR activity, which can be reversed by various inverse agonists. The β-agonist-induced changes could represent a novel regulation mechanism of β2-AR activity.