Cell Surface Binding, Intracellular cAMP Elevation and Membrane Associated G-Protein Activation by (R,R)- and (S,S)-Formoterols in Androgen Independent Human Prostate Cancer Cell Line, PC3

The commercially available long acting anti-asthmatic drug, formoterol exists as a racemate of four enantiomers ((R,R)-, (R,S)-, (S,R)- and (S,S)-. The study describes several comparisons between two completely different enantiomers, (R,R)- and (S,S)- based on their # 1) cell surface binding, # 2) cAMP elevation ability, # 3) G-protein activation and # 4) inhibition of DNA synthesis in PC3 cells. The presence of high affinity β₂-adrenergic receptor (K_d ∼ 30 pmol/L) was confirmed by competition binding of ¹²⁵I-cyanopindolol with increasing concentration of (R,R)-formoterol using both intact PC3 cells and isolated plasma membrane. Replacing (R,R)- by (S,S)- yielded no significant binding interaction proving its ineffectiveness toward the β₂-adreno-receptor. While both were capable of eliciting prolonged cAMP generating activity in intact PC3 cells, the EC₅₀ values (R,R- = 10.5 pM, R,S- = 11.0 pM and S,S = 1000 pM) varied nearly 100 fold in favor of (R,R)- and (R,S)-. Propanolol effectively inhibited cAMP elevation in intact cells in both the cases as also agonist stimulated incorporation of [γ³²P]- GTP-AA (Guanosine triphosphate azidoanilide) by (R,R)- in isolated PC3 membrane, but failed in both events in the presence of (S,S)- enantiomer although incorporation of [γ³²P]-GTP-AA is specific for both the enantiomers. The unique discriminatory behavior is further observed in presence of muscarinic agonist, carbachol, which potentiated cAMP generation by (R,R)- nearly 2-3 fold, but was unable to do so in the presence of (S,S)-. These facts confirmatively indicate that cAMP elevation by (S,S)- in PC3 cells occurs entirely via a different pathway than its (R,R)- counterpart. Interestingly, both enantiomers can effectively lower the DNA synthesis, showing superior efficacy of (R,R)-.