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Background: b2-adrenoceptor agonists are effective bronchodilators. In vitro studies demonstrated long-lasting airway smooth muscle relaxation by salmeterol after washout, the quick disappearance of this effect in presence of antagonists and its recovery after antagonist removal. Current explanations invoke salmeterol accumulation in the membrane ('diffusion microkinetic' model) or the existence of salmeterol-binding 'exosites'. An alternative model based on 'rebinding' of a dissociated ligand to the receptor molecules also produces an apparent decrease in the ligand's dissociation rate in the absence of competing ligands. Purpose and approach: Computer-assisted simulations were performed to follow the receptor-occupation by a salmeterol-like ligand and a competing ligand as a function of time. The aptness of the models to describe the above in vitro findings was evaluated. Key results: The 'diffusion microkinetic' model is sufficient to explain a long-lasting b2-adrenoceptor stimulation and reassertion as long as the membrane harbors a high concentration of the agonist. At lower concentration, 'rebinding' and, in second place, 'exosite' binding are likely to become operational. Conclusions and implications:The 'rebinding' and 'exosite' binding mechanisms take place at a sub-cellular/molecular scale. Pending their demonstration by experiments on appropriate, simple models such as intact cells or membranes thereof, these mechanisms remain hypothetical in the case of salmeterol. Airway smooth muscle contraction could also be governed by additional mechanisms that are particular to this macroscopic approach.

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BACKGROUND AND PURPOSEBecause the in vivo effectiveness of ligands may also be determined by the rate by which they dissociate from their target receptors, drug candidates are being increasingly screened for this kinetic property. The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow the association of subsequently added radioligand molecules. EXPERIMENTAL APPROACHWe used the 'two-step competition' binding approach consisting of pre-incubating the receptor preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time. Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass action to a single class of sites, the unlabelled ligand's disociation rate can be estimated from the upward shift that the competition curve experiences after washing. KEY RESULTSThe relevance of the 'two-step competition' approach was explored by computer simulations and by comparing the dissociation behaviour of unlabelled D2 dopamine and CB1 cannabinoid receptor antagonists in this and alternative approaches. Besides providing satisfactory estimations of dissociation rates, the method also detects the ability of the unlabelled ligand molecules to be released from 'sinks' such as the cell membrane. CONCLUSIONS AND IMPLICATIONSAs the 'two-step competition' requires rapid intermediate washing steps and needs radioligand binding to be measured at only one time point, this approach is particularly suited for binding studies on intact plated cells. LINKED ARTICLESThis article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi. org/10.1111/bph.2010.161.issue-6 Abbreviations CHO-D2S cells, recombinant Chinese hamster ovary cells stably expressing the human D2S dopamine receptor; HEK293-CB1r cells, recombinant human embryonic kidney cells stably expressing human Cannabinoid CB1 receptor; IC50, concentration at which the competitor produces half-maximal inhibition of specific radioligand binding; k4, dissociation rate constant of an unlabelled competitor; TSC, two-step competition

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