FUNCTIONAL-ACTIVITY OF ANTIBODIES AT THE BOVINE BETA(2)-ADRENOCEPTOR

Antibodies that can activate beta(2)-adrenoceptors (beta(2)-AR) have t he potential to mimic the anabolic effects of beta-agonist drugs, such as clenbuterol. In this study, antibodies were raised in rabbits agai nst two peptide analogues of the human beta(2)-adrenoceptor(beta(2)-AR ): One peptide corresponded to the complete second outer loop of the r eceptor (24 amino acids; H24T), and the second peptide was a truncated version of the first (13 amino acids; H13C). Following affinity purif ication, the antibodies were screened to detect interaction with beta( 2)-AR in vitro. Membrane proteins from transformed Escherichia coli th at express the beta(2)-AR were separated using SDS PAGE and transferre d to nitrocellulose sheets. Immunoblotting revealed a single protein b and (39,000 Da) that was recognized by the affinity-purified anti-H24T antibodies. However, the anti-H13C antibodies did not recognize any p rotein bands in immunoblots. In ligand binding studies, anti-H24T anti bodies at a concentration of 50 nM, increased the affinity (KD) of the radiolabeled antagonist [I-125]iodocyanopindolol for the bovine beta( 2)-AR from 31.7 pM to 25.3 pM (P < .05) without changing the receptor number. Anti-H13C antibodies had no effect on ligand binding. In compe titive ligand binding experiments, there was no effect of antibodies o n the affinity of bovine beta(2)-AR for the agonist (-)-isoproterenol. However, functional activity of anti-H24T antibodies was demonstrated in an organ bath study. The presence of antibodies caused a leftward shift in the concentration-response curve for (-)-isoproterenol-induce d relaxation of isolated bovine smooth muscle strips. Values for pD(2) (-log EC50) were reduced in the presence of 10 nM antibody (8.62 +/- .11) compared to controls (8.30 +/- .08; P < .05). Anti-H13C antibodie s had no effect on (-)-isoproterenol-induced smooth muscle relaxation. These studies have demonstrated recognition, interaction, and functio nal activity of site-directed antibodies at the beta(2)-AR. Further st udies will determine whether antibodies that potentiate activity at th e beta(2)-AR may be evoked by the active immunization of cattle with t he peptide H24T, and if so, whether this will cause the repartitioning of nutrients in a manner analogous to conventional beta(2)-agonists a nd thus provide an alternative to the use of xenobiotic compounds.