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 injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) during the induction phase, and β ₂-adrenoceptor (β ₂-AR), G_s alpha subunit (Gα _s), and protein kinase A (PKA) involvement in subsequent cardioprotection. Here, we tested for induction of a protective β ₂-AR/Gα _s/PKA signaling axis with SLP to ascertain whether signaling changes were PI3K-dependent (by sustained cotreatment 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 (using morphine) in association with increased membrane versus reduced cytosolic levels of total and phosphorylated β ₂-ARs; increased membrane and cytosolic expression of 52 and 46 kDa Gα _s isoforms, respectively; and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of β ₂-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol, and 6-[3-(dimethylamino)propionyl]-forskolin. Protection with SLP was not modified by ROCK inhibition, and changes in β ₂-AR, Gα _s, and PKA expression appeared insensitive to PI3K inhibition, although 5 days of wortmannin alone exerted unexpected effects on signaling (also increasing membrane β ₂-AR and PKA expression/phosphorylation and Gα _s levels). In summary, sustained OR agonism upregulates cardiac membrane β ₂-AR expression and phosphorylation in association with increased Gα _s subtype levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears nonessential to OR upregulation of the β ₂-AR signal axis. This opioidergic remodeling of β ₂-AR signaling may explain β ₂-AR, Gα _s, and PKA dependence of SLP protection.