Kinetic analysis of drug-receptor interactions of long-acting beta(2), sympathomimetics in isolated receptor membranes: Evidence against prolonged effects of salmeterol and formoterol on receptor-coupled adenylyl cyclase

The long-acting beta(2) sympathomimetics salmeterol and formoterol have bee n presumed to exert their prolonged action either by binding to an accessor y binding site ("exo-site") near the beta(2) adrenoceptor or by their high affinity for beta(2) adrenoceptors and correspondingly slow dissociation. W hereas most studies with salmeterol had been done in intact tissues, which have slow diffusion and compartmentation of drugs in lipophilic phases, tha t restrict drug access to the receptor biophase, we used purified receptor membranes from rat lung and disaggregated calf tracheal myocytes as model s ystems. Binding experiments were designed to measure the slow dissociation of agonists by means of delayed association of (-)-[I-125]iodopindolol. Rat lung membranes were pretreated with high concentrations of agonists (salme terol, formoterol, isoprenaline) before dissociation was induced by 50-fold dilution. Half-times of association of (-)-[I-125]iodopindolol remained un changed compared with untreated controls, indicating that dissociation of a gonists occurred in less than 2 min. Adenylyl cyclase experiments were desi gned to determine the on and off kinetics of agonists to beta(2) adrenocept ors by measuring the rate of receptor-induced cyclic AMP (cAMP) formation. Experiments were performed in tracheal membranes characterized by high V-ma x values of cAMP formation. Adenylyl cyclase activation occurred simultaneo usly with the addition of the agonist, continued linearly with time for 60 min, and ceased immediately after the antagonist was added. Similarly, when receptor membranes were preincubated in a small volume with high salmetero l concentrations, there was a linear increase in cAMP formation, which was immediately interrupted by a 100-fold dilution of the reaction mixture. Thi s militates against the exo-site hypothesis. On the other hand, dissociatio n by dilution was much less when membranes were preincubated with a large v olume of salmeterol at the same concentration. indicating that physicochemi cal effects, and not exo-site binding, underlie its prolonged mode of actio n.