title: Contribution of the extracellular cAMP-adenosine pathway to dual coupling of β2-adrenoceptors to Gs and Gi proteins in mouse skeletal muscle.
abstract: 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.
authors.affiliation: Division of Cellular Pharmacology, Department of Pharmacology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100 São Paulo, SP Brazil 04044-020.
authors.forename: Thiago
authors.initials: T
authors.lastname: Duarte
authors.forename: Francisco Sandro
authors.initials: FS
authors.lastname: Menezes-Rodrigues
authors.forename: Rosely Oliveira
authors.initials: RO
authors.lastname: Godinho
mesh_terms: D000241:Adenosine / Q000502:physiology*; D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002976:Clenbuterol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D003964:Diaphragm / Q000187:drug effects / Q000378:metabolism*; D004558:Electric Stimulation; D025461:Feedback, Physiological / Q000502:physiology; D005280:Fenoterol / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D008297:Male; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D020889:Rolipram / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D005576:Colforsin; D005280:Fenoterol; D000242:Cyclic AMP; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D020889:Rolipram; D000241:Adenosine; D002976:Clenbuterol