Dissociations in the Effects of β2-Adrenergic Receptor Agonists on cAMP Formation and Superoxide Production in Human Neutrophils: Support for the Concept of Functional Selectivity

In neutrophils, activation of the β 2 -adrenergic receptor (β 2 AR), a G s -coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β 2 AR ligands on adenosine-3′,5′-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O 2 •− ) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O 2 •− formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β 2 AR agonists were generally more potent in inhibiting fMLP-induced O 2 •− production than in stimulating cAMP accumulation. (−)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O 2 •− production. Moreover, (−)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O 2 •− assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O 2 •− production after β 2 AR-stimulation although cAMP-increasing substances inhibited O 2 •− production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β 2 AR inhibits O 2 •− production in a cAMP-independent manner.