Binding of (-)-[3H]-CGP12177 at two sites in recombinant human beta 1-adrenoceptors and interaction with beta-blockers.
To verify the hypothesis that the non-conventional partial agonist (-)-CGP12177 binds at two beta(1)-adrenoceptor sites, human beta(1)-adrenoceptors, expressed in CHO cells, were labelled with (-)-[(3)H]-CGP12177. We compared the binding affinity and antagonist potency of 12 clinically used beta-blockers against the cyclic AMP-enhancing effects of (-)-isoprenaline and (-)-CGP12177.(-)-[(3)H]-CGP12177 bound to a high affinity site (H; K(H)=0.47 nM) and low affinity site (L); K(L)=235 nM). (-)-[(3)H]-CGP12177 dissociated from the beta(1)-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 -logEC(50)M of 8.2 and 7.6. (-)-CGP12177 antagonised the effects of (-)-isoprenaline with a pK(B) of 9.9. The beta-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 beta-blockers, estimated from competition with 3-5 nM (-)-[(3)H]-CGP12177 (binding to the H-site), correlated with the corresponding affinities estimated from antagonism of the (-)-isoprenaline effects. We conclude that (-)-[(3)H]-CGP12177 binds at two sites in the recombinant beta(1)-adrenoceptor. (-)-CGP12177 is an antagonist of catecholamine effects through the H-site and a non-conventional partial agonist through the L-site. beta-blockers are more potent antagonists through the H-site than the L-site.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T2 T30 T31 T32 T33 T34 T35 T36 T37 T3 T4 T5 T6 T7 T8 T9
CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
(+/-)-bisoprolol (+/-)-alprenolol (+/-)-nadolol (-)-bupranolol (-)-[(3)H]-CGP12177 (-)-isoprenaline (-)-[(3)H]-CGP12177 (-)-CGP12177 catecholamine (-)-[(3)H]-CGP12177 (-)-CGP12177 cyclic AMP (-)-isoprenaline (-)-CGP12177 (-)-[(3)H]-CGP12177 (-)-[(3)H]-CGP12177 (-)-CGP12177 (-)-Isoprenaline (-)-CGP12177 cyclic AMP (-)-CGP12177 (-)-atenolol (-)-isoprenaline (-)-isoprenaline (-)-[3H]-CGP12177 human beta(1)-adrenoceptors beta(1)-adrenoceptor beta(1)-adrenoceptors beta(1)-adrenoceptor human beta 1-adrenoceptors (+/-)-metropolol (-)-pindolol (-)-timolol (+/-)-carvedilol (+/-)-oxprenolol (+/-)-sotalol (-)-propranolol