To verify the hypothesis that the non-conventional partial agonist (−)-CGP12177 binds at two β₁-adrenoceptor sites, human β₁-adrenoceptors, expressed in CHO cells, were labelled with (−)-[³H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used β-blockers against the cyclic AMP-enhancing effects of (−)-isoprenaline and (−)-CGP12177.

(−)-[³H]-CGP12177 bound to a high affinity site (H; K_H=0.47 nM) and low affinity site (L); K_L=235 nM). (−)-[³H]-CGP12177 dissociated from the β₁-adrenoceptors with a fast component (k_off=0.45 min⁻¹), consistent with the L-site, and a slow component (k_off=0.017–0.033 min⁻¹), consistent with the H-site. (−)-Isoprenaline and (−)-CGP12177 caused 96-fold and 12-fold maximal increases in cyclic AMP levels with −logEC₅₀M of 8.2 and 7.6. (−)-CGP12177 antagonised the effects of (−)-isoprenaline with a pK_B of 9.9. The β-blockers antagonised the effects of (−)-isoprenaline more than the effects of (−)-CGP12177 with potency ratios: (−)-atenolol 1,000, (±)-metropolol 676, (−)-pindolol 631, (−)-timolol 589, (±)-carvedilol 204, (±)-oxprenolol 138, (±)-sotalol 132, (−)-propranolol 120, (±)-bisoprolol 95, (±)-alprenolol 81, (±)-nadolol 68 and (−)-bupranolol 56. In intact cells the binding constants of β-blockers, estimated from competition with 3–5 nM (−)-[³H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (−)-isoprenaline effects.

We conclude that (−)-[³H]-CGP12177 binds at two sites in the recombinant β₁-adrenoceptor. (−)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. β-blockers are more potent antagonists through the H-site than the L-site.