Beta 2-adrenoceptor activation by zinterol causes protein phosphorylation, contractile effects and relaxant effects...

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both beta 1-adrenoceptors (beta 1AR) and beta 2-adrenoceptors (beta 2AR) mediate positive inotropic effects but that only beta 1AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both beta 1AR and beta 2AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose beta-AR comprise around 2/3 of beta 1AR and 1/3 of beta 2AR, whether or not beta 2AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate beta 2AR, we used the beta 2AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t1/2 of relaxation) effects with EC50 values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the beta 1AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the beta 2AR-selective antagonist ICI 118551 (50 nM) which reduced both EC50 values to 1 microM. Zinterol stimulated adenylyl cyclase activity with an EC50 of 30 nM and intrinsic activity of 0.75 with respect to (-)-isoprenaline (600 microM); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane beta AR labelled with (-)-[125I] cyanopindolol with higher affinity for beta 2AR than for beta 1AR; the binding to beta 2AR but not to beta 1AR was reduced by GTP gamma S (10 microM). In the presence of CGP 20712A (300 nM) (-)-isoprenaline (400 microM) (to activate both beta 1AR and beta 2AR maximally) and zinterol (10 microM) increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation t1/2 by 32% and 18% respectively. These effects of (-)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 +/- 2.0 vs 12.4 +/- 2.3 and 10.1 +/- 2.5 vs 8.6 +/- 1.6 respectively. (-)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 +/- 0.3 vs 0.4 +/- 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both beta 1AR and beta 2AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.