Regulation of the β-adrenergic receptor signaling pathway in sustained ligand-activated preconditioning

Sustained ligand-activated preconditioning (SLP), induced with chronic opioid receptor (OR) agonism, enhances tolerance to ischemia/reperfusion (I/R) injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate PI3K-Akt during the induction phase, and β2-adrenoceptor (β2-AR), Gαs and protein kinase A (PKA) involvement in subsequent cardioprotection. Here we tested for induction of a protective β2-AR-Gαs-PKA signaling axis with SLP, whether signaling changes were PI3K-dependent (by sustained co-treatment with wortmannin), and whether the downstream PKA target Rho-kinase (ROCK) participates in subsequent cardioprotection (by acute treatment with fasudil). A protected phenotype was evident after 5 days of OR agonism, in association with increased membrane vs. reduced cytosolic levels of total and phosphorylated β2-ARs, increased membrane and cytosolic expression of 52 and 46 kDa Gαs isoforms, respectively, and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of β2-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol and NKH477. Protection with SLP was not modified by ROCK inhibition, and changes in β2-AR, Gαs and PKA expression appeared insensitive to PI3K inhibition, although 5 day wortmannin alone exerted unexpected effects on signaling (also increasing membrane β2-AR and PKA expression/phosphorylation, and Gαs levels). In summary, sustained OR agonism up-regulates cardiac membrane β2-AR expression and phosphorylation in association with increased Gαs sub-type levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears non-essential to OR up-regulation of the β2-AR signal axis. This opioidergic remodeling of β2-AR signaling may explain β2-AR, Gαs and PKA dependence of SLP protection.