β₂-Adrenoceptor activation by zinterol causes protein phosphorylation, contractile effects and relaxant effects through a cAMP pathway in human atrium

Abstract

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both β₁-adrenoceptors (β₁AR) and β₂-adrenoceptors (β₂AR) mediate positive inotropic effects but that only β₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both β₁ AR and β₂ AR 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 β-AR comprise around 2/3 of β₁AR and 1/3 of β₂AR, whether or not β₂AR-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 β₂AR, we used the β₂AR-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 t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the β AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the β₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 μM. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (−)-isoprenaline (600 μM); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (−)-[¹²⁵I] cyanopindolol with higher affinity for β₂AR than for β- 1 AR; the binding to β₂AR but not to β- BAR was reduced by GTPyS (10 μM). In the presence of CGP 20712A (300 nM) (−)-isoprenaline (400 μM); (to activate both β₁AR and β₂AR maximally) and zinterol (10 μM); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut 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 β₁AR and β₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.