β2-adrenoceptor (β2-AR) agonists increase skeletal muscle contractile force via activation of Gs protein/ adenylyl cyclases (AC) and increased generation of cAMP. Herein, we evaluated the possible dual coupling of β2-AR to Gs and Gi proteins and the influence of β2-AR/Gs-Gi/cAMP signaling cascade on skeletal muscle contraction. Assuming that increment of intracellular cAMP is followed by cAMP efflux and extracellular generation of adenosine, the contribution of 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-Br-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, 20 μM rolipram induced transient positive inotropic effects that peaked 30 min after stimulation onset, declining to 10-20% of peak levels in 30 min. The late descending phase of β2-AR agonist inotropic effect was mimicked by either cAMP or adenosine and abolished by preincubation of diaphragm with pertussis toxin (PTX, Gi signaling inhibitor) or the organic anion transporter inhibitor probenecid, indicating a delayed coupling of β2-AR to Gi protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β2-AR inotropic effect was inhibited by the A1 adenosine receptor antagonist DPCPX and ecto-5'-phosphodiesterase inhibitor AMPCP, indicating that β2-AR coupling to Gi is indirect and dependent on A1 receptor activation. The involvement of extracellular cAMP-adenosine pathway in β2-AR signaling would provide a negative-feedback loop that may limit GsPCR positive inotropism and potential deleterious effects of excessive contractile response.
- Received February 8, 2012.
- Revision received March 19, 2012.
- Accepted March 20, 2012.
- The American Society for Pharmacology and Experimental Therapeutics