Abstract
The long-acting β2 sympathomimetics salmeterol and formoterol have been presumed to exert their prolonged action either by binding to an accessory binding site (“exo-site”) near the β2 adrenoceptor or by their high affinity for β2 adrenoceptors and correspondingly slow dissociation. Whereas most studies with salmeterol had been done in intact tissues, which have slow diffusion and compartmentation of drugs in lipophilic phases, that restrict drug access to the receptor biophase, we used purified receptor membranes from rat lung and disaggregated calf tracheal myocytes as model systems. Binding experiments were designed to measure the slow dissociation of agonists by means of delayed association of (−)-[125I]iodopindolol. Rat lung membranes were pretreated with high concentrations of agonists (salmeterol, formoterol, isoprenaline) before dissociation was induced by 50-fold dilution. Half-times of association of (−)-[125I]iodopindolol remained unchanged compared with untreated controls, indicating that dissociation of agonists occurred in less than 2 min. Adenylyl cyclase experiments were designed to determine the on and off kinetics of agonists to β2 adrenoceptors by measuring the rate of receptor-induced cyclic AMP (cAMP) formation. Experiments were performed in tracheal membranes characterized by highVmax values of cAMP formation. Adenylyl cyclase activation occurred simultaneously 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 salmeterol concentrations, there was a linear increase in cAMP formation, which was immediately interrupted by a 100-fold dilution of the reaction mixture. This militates against the exo-site hypothesis. On the other hand, dissociation by dilution was much less when membranes were preincubated with a large volume of salmeterol at the same concentration, indicating that physicochemical effects, and not exo-site binding, underlie its prolonged mode of action.
Footnotes
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Send reprint requests to: Prof. Dr. H. Lemoine, University of Düsseldorf, Institute for Laser Medicine, Molecular Drug Research Group, Universitätsstr. 1, 40 225 Düsseldorf, Germany. E-mail: lemoine{at}uni-duesseldorf.de
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↵1 This work was supported by the Deutsche Forschungsgemeinschaft (DFG, LE 552-1).
- Abbreviations:
- AC
- adenylyl cyclase
- BU
- (−)-bupranolol
- CGP 20
- 712 A, 1-[2(3-carbamoyl-4-hydroxy phenoxy)-ethylamino]3-[4-(1-methyl-4-trifluoromethyl-2 imidazolyl)phenoxy]2-propanol methanesulfonate
- FOR
- (−)-formoterol
- Gpp(NH)p
- 5′-guanylylimidodiphosphate
- IA
- intrinsic activity
- ISO
- (−)-isoprenaline
- SLM
- (±)-salmeterol
- AR
- adrenoceptors
- SYM
- sympathomimetics
- IPIN
- (−)-iodopindolol
- Received June 30, 1998.
- Accepted September 28, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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