Abstract

β₂-Adrenoceptor (β₂-AR) agonists increase skeletal muscle contractile force via activation of G_s protein/adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β₂-AR to G_s and G_i proteins and the influence of the β₂-AR/G_s-G_i/cAMP signaling cascade on skeletal muscle contraction. Assuming that the increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of the extracellular cAMP-adenosine pathway on the β₂-AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β₂-AR agonists), forskolin (AC activator), cAMP/8-bromo-cAMP, and adenosine were evaluated on isometric contractility of mouse diaphragm muscle induced by supramaximal direct electrical stimulation (0.1 Hz, 2 ms duration). Clenbuterol/fenoterol (10–1000 μM), 1 μM forskolin, and 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10 to 20% of peak levels in 30 min. The late descending phase of the β₂-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX) (G_i signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β₂-AR to G_i protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β₂-AR inotropic effect was inhibited by the A₁ adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5′-phosphodiesterase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt, indicating that β₂-AR coupling to G_i is indirect and dependent on A₁ receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β₂-AR signaling would provide a negative feedback loop that may limit stimulatory G protein-coupled receptor positive inotropism and potential deleterious effects of excessive contractile response.