Onset of β2 adrenoceptor agonist signalling in primary human bronchial smooth muscle cells is correlated with intrinsic efficacy

Elizabeth M Rosethorne, Robert J Turner, Steven J Charlton

Novartis Institutes for Biomedical Research, Horsham, UK

Inhaled β2 adrenergic receptor (β2 AR) agonists are widely used as bronchodilator therapies for asthma and COPD. Different agonists have varying rates of onset of action, e.g. indacaterol and salbutamol are effective bronchodilators after 5 min whereas salmeterol takes 15 min to achieve significant bronchodilation over baseline (Brookman et al, 2007). This has been attributed to differences in the lipophilicity of the agonists i.e hydrophobic ligands take longer to diffuse into tissue and may even access the receptor via the membrane compartment (Anderson et al, 1994). While this holds true for salmeterol and salbutamol, the relatively high lipophilicity of indacaterol should result in a slower onset of action. We have recently demonstrated a correlation between agonist efficacy and the rate of β-arrestin recruitment to the β2 AR (Rosethorne & Charlton, 2008), with high efficacy ligands generating a faster rate of recruitment. Here we have explored this phenomenon further, characterizing efficacy and rate of cAMP accumulation in primary human bronchial smooth muscle cells (hBSMCs).

hBSMCs were grown to confluency in 96-well OptiPlates, before incubation with agonist or vehicle for 1 h at room temperature in HBSS supplemented with 5 mM HEPES and 0.1 % bovine serum albumin. cAMP levels were assessed using AlphaScreen reagents, read on a Packard EnVision plate reader. For time-course studies, hBSMCs were grown to confluence in 12-well plates and prelabeled with 4 μCi/ml of [2, 8-3H]adenine (PerkinElmer). Cells were stimulated with a range of β2 AR agonists (at EC80) for between 5 and 30 min. [3H]-cAMP was then isolated by sequential Dowex and alumina chromatography, and quantified using scintillation counting. Results are shown in the table below, data are expressed as mean ± s.e. mean (n ≥ 3).

There was a good correlation between rate of cAMP generation and intrinsic activity for salmeterol, indacaterol and formoterol (r2 = 0.996). Salbutamol, however, appeared to have a faster rate of signalling than would be predicted from its efficacy. This may reflect the high degree of hydrophilicity of salbutamol when compared to the other agonists, allowing more rapid diffusion to the receptor. In conclusion, we have demonstrated that the rate of cAMP accumulation is influenced by agonist efficacy and that this, in combination with lipophilicity, may explain why β2 AR agonists demonstrate differences in their onset of action.

Anderson GP, et al., Lindén A, Rabe KF (1994) Eur Respir J. 7:569-78

Brookman et al., (2007) Current Medical Research and Opinion 23:3113

Rosethorne, E & Charlton, SJ (2008) Focused Meeting: High Throughput Pharmacology 2008