Abstract
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 the β2-AR/Gs-Gi/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) (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 8-cyclopentyl-1,3-dipropylxanthine and ecto-5′-phosphodiesterase inhibitor α,β-methyleneadenosine 5′-diphosphate sodium salt, indicating that β2-AR coupling to Gi 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.
Footnotes
This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo and Conselho Nacional de Desenvolvimento Científico e Tecnológico (to R.O.G.). F.S.M.-R. was an MSc fellow from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and T.D. was an MSc fellow from Conselho Nacional de Desenvolvimento Científico e Tecnológico.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- β-AR
- β-adrenoceptor
- GPCR
- G protein-coupled receptor
- AC
- adenylyl cyclase
- MRP
- multidrug resistance protein
- PDE
- phosphodiesterases
- CGS-15943
- 5-amino-9-chloro-2-(2-furyl)1,2,4-trizolo[1,5-c]quinazoline
- DPCPX
- 8-cyclopentyl-1,3-dipropylxanthine
- 8-Br-cAMP
- 8-bromo-cAMP
- ADO
- adenosine
- AMPCP
- α,β-methyleneadenosine 5′-diphosphate sodium salt
- PTX
- pertussis toxin
- ANOVA
- analysis of variance.
- Received February 8, 2012.
- Accepted March 20, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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