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Contribution of the Extracellular cAMP-Adenosine Pathway to Dual Coupling of β 2 -Adrenoceptors to G s and G i Proteins in Mouse Skeletal Muscle

2012; American Society for Pharmacology and Experimental Therapeutics; Volume: 341; Issue: 3 Linguagem: Inglês

10.1124/jpet.112.192997

1521-0103

Thiago Duarte, Francisco Sandro Menezes Rodrigues, Rosely Oliveira Godinho,

Adipose Tissue and Metabolism

β 2 -Adrenoceptor (β 2 -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 β 2 -AR to G s and G i proteins and the influence of the β 2 -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 β 2 -AR inotropic response was also addressed. The effects of clenbuterol/fenoterol (β 2 -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 β 2 -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 β 2 -AR to G i protein which depends on cAMP efflux. Remarkably, the PTX-sensitive β 2 -AR inotropic effect was inhibited by the A 1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine and ecto-5′-phosphodiesterase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt, indicating that β 2 -AR coupling to G i is indirect and dependent on A 1 receptor activation. The involvement of the extracellular cAMP-adenosine pathway in β 2 -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.