A cell-based assay to measure the duration of action of ß2 adrenoreceptor agonists at the human receptor

Susan Summerhill, Tim Stroud, Mike Trevethick & Christelle Perros-Huguet. Allergy & Respiratory Biology, Pfizer Global R&D, Sandwich, UK.

Current in-vitro methods for determining duration of action of ß2 adrenoreceptor agonists typically involve guinea-pig isolated trachea strips in organ bath systems (Coleman et al 1989 and 1996) and are often labour intensive and time consuming. We have developed and characterised a cell-based "wash off" assay to assess the duration of action of ß2 adrenoreceptor agonists in Chinese Hamster Ovary cells recombinantly expressing the human ß2 adrenoreceptor (CHOh ß2).

CHOh ß2 cells were seeded in 96-well Viewplates (Perkin Elmer) at 0.2x106 cells/well in 1% FBS/DMEM-F12 over night at 37oC. Concentration effect curves to ß2 adrenoreceptor agonists following a 30-minute incubation at 37oC were constructed by measuring increases in intracellular cAMP levels using a HitHunter cAMP IITM assay (DiscoveRx). EC50 values (concentration of agonist needed to produce half the maximal response to that agonist) for cells stimulated with agonist (unwashed) were compared to EC50 values generated in cells exposed to agonists for 30 minutes, washed with PBS and incubated in agonist free media for a further 30 minutes prior to cAMP determination (washed). For ease of comparison, unwashed and washed EC50 values for each compound were used to generate a fold rightward shift (RWS) on washing (RWS = EC50 washed / EC50 unwashed).

The ß2 adrenoreceptor agonists caused a concentration related increase in intracellular cAMP with rank order of potency: Formoterol > Salmeterol > Isoprenaline > Salbutamol (see Table 1). On washing, the EC50 values of the short-acting ß2 agonists Salbutamol and Isoprenaline shifted significantly (p< 0.01) to the right, whereas the longer-acting ß2 agonists Formoterol and Salmeterol produced smaller shifts indicating they were more resistant to washing.

Table 1 .Comparison of Unwashed and Washed EC50 curves. Data are geometric mean and 95% confidence interval values from n=4-9, ap<0.01 difference between washed and unwashed EC50, bp<0.01 RWS different from Salmeterol (using ANOVA).

Salbutamol, Isoprenaline, Formoterol and Salmeterol produce statistically distinct washout profiles suggesting that this cell-based assay could be used to rank novel ß2 adrenoreceptor agonists on their potency and duration of action prior to any tissue or in-vivo determinations. Further experiments are underway to try and determine if the long duration of action is due to “exosite” binding, slow receptor offset or membrane affinity.

Coleman RA et al. (1989) J. Pharmacol Methods, 21: 71-86.
Coleman RA et al. (1996) Pulm. Pharmacol., 9: 107-117.