Kinetic analysis of endogenous β₂ -adrenoceptor-mediated cAMP GloSensor™ responses in HEK293 cells

Standard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands. However, the occurrence of non-equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to β₂ -adrenoceptor agonists in HEK293 cells expressing the endogenous β₂ -adrenoceptor at extremely low levels.

The kinetics of β₂ -adrenoceptor agonist-stimulated cAMP responses were monitored in real-time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensor™ biosensor. Potency (EC₅₀ ) and efficacy (E_max ) values were determined at the peak of the agonist GloSensor™ response and compared to kinetic parameters L₅₀ and IR_max values derived from initial response rates.

The partial agonists salbutamol and salmeterol displayed reduced relative IR_max values (with respect to isoprenaline) when compared with their E_max values. Except for the fast dissociating bisoprolol, preincubation with β₂ -adrenoceptor antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi-equilibrium in this low receptor reserve system. This effect was exacerbated when IR_max parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IR_max consistent with its short residence time.

Kinetic analysis of real-time signalling data can provide valuable insights into the hemi-equilibria that can occur in low receptor reserve systems with agonist-antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing.