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.
A key physiological mechanism to increase skeletal muscle contractile force relies on circulating hormones that elevate intracellular cAMP. Endogenous sympathomimetic amines or synthetic β-adrenoceptor (β-AR) agonists, such as nonselective isoproterenol (Bowman and Nott, 1969; Andrade-Lopes et al., 2011) or β2-AR-selective salbutamol (Easton et al., 2008) increase muscle contraction force via activation of stimulatory G protein (Gs)-coupled receptors (GPCRs), which in turn activates adenylyl cyclases (ACs). The sequential increase in intracellular cAMP (Bowman and Nott, 1969; Cairns and Dulhunty, 1993; Andrade-Lopes et al., 2011) and activation of protein kinase A result in phosphorylation of ryanodine receptors and increased Ca2+ release from the sarcoplasmic reticulum (Reiken et al., 2003; Lynch and Ryall, 2008), which culminates in potentiation of muscle contraction. However, in many cells, β-AR/Gs/AC-mediated responses are affected by promiscuous coupling of β-AR to other Gα subunits, especially with the inhibitory Gαi subfamily as reported in Sf9 insect cells overexpressing β2-AR (Wenzel-Seifert and Seifert, 2000) and human embryonic kidney 293 cells (Daaka et al., 1997). In addition, secondary coupling of β2-AR to Gi proteins in rodent (Kuschel et al., 1999; Xiao et al., 1999) and human (Kilts et al., 2000) heart muscle cells has been associated with a cardioprotective effect of β2-AR agonists, via attenuation of Gs-mediated inotropic response.
In fact, dual coupling of β2-AR with Gs and Gi proteins has already been reported in rat skeletal muscle (Gosmanov et al., 2002) and in the L6 myogenic cell line (Nevzorova et al., 2006). However, the precise pathways implicated in this signaling shift and its physiological significance on regulation of skeletal muscle contraction remain unknown.
In skeletal muscle, the β-AR-dependent increase of intracellular cAMP is followed by the efflux of cyclic nucleotide via probenecid-sensitive transporters (Godinho and Costa, 2003). Within the large superfamily of ATP-binding cassette transporters, three members of the subfamily of multidrug resistance protein (MRP), MRP4, MRP5, and MRP8, are able to transport cAMP (for review, see Sager and Ravna, 2009), and two of them, MRP4 and MRP5, are expressed in skeletal muscle (Knauer et al., 2010).
Once outside the muscle fiber, cAMP is sequentially metabolized to AMP and adenosine by ecto-phosphodiesterases (PDEs) and ecto-nucleotidases (Chiavegatti et al., 2008). Considering this fact and the expression of Gi-coupled A1 and A3 adenosine receptors in skeletal muscle tissue (Lynge and Hellsten, 2000; Zheng et al., 2007), we suggest that indirect coupling of β2-AR to Gi protein via activation of adenosine receptors would have physiological significance. Our working hypothesis is that the extracellular adenosine, generated as a consequence of activation of β2-AR/Gs/AC signaling, may influence the initial positive inotropic response by autocrine stimulation of adenosine receptors coupled to Gi protein.
Thus, in the present study, we have used in vitro mouse diaphragm preparation to evaluate the effect of continuous β2-AR activation on skeletal muscle contraction, focusing on its coupling to distinct G proteins and the possible influence of the extracellular cAMP-adenosine pathway on β2-AR-mediated positive inotropic response.
Materials and Methods
All animal procedures were approved by the Institutional Research Ethics Committee (protocols 0011/08 and 0072/11) at Universidade Federal de São Paulo and have been performed in accordance with the Declaration of Helsinki and/or with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) as adopted and promulgated by the National Institutes of Health. All experiments were performed using diaphragm strips isolated from 90- to 120-day-old male Swiss mice from the institutional animal care facility.
Isometric Contraction of Mouse Diaphragm Muscle.
Mice were killed by cervical dislocation, and diaphragm strips were prepared according to Souccar et al. (2005) with modifications (Andrade-Lopes et al., 2011). In brief, diaphragm strips with rib were quickly dissected and placed in Tyrode's solution (135 mM NaCl, 5 mM KCl, 1 mM MgCl2 · 6 H2O, 15 mM NaHCO3, 2 mM NaH2PO4 · H2O, 2 mM CaCl2 · 2 H2O, and 11 mM glucose, pH 7.4). The rib was gently pinned to a holder fixed in the bottom of a organ bath filled with 2 ml of Tyrode's solution and a segment of central diaphragm tendon attached to a PowerLab force transducer (ADInstruments, Sydney, Australia), which was maintained at 30°C and continuously gassed with 95% O2/5% CO2. Isometric twitch contraction was elicited by electrical stimulation of muscle strips through silver electrodes, with 0.1 Hz frequency, 2 ms duration, and supramaximal voltage (Grass Stimulator S88; Grass Technologies, West Warwick, RI), under optimal tension. d-Tubocurarine (10 μM) was added to the solution to block neuromuscular transmission and prevent double stimulation of the muscle from the remaining axon stub. After a 20- to 30-min stabilization, muscle length was readjusted to give an optimal twitch tension, and 30 min later the effect of drugs was investigated, as described below. Data were collected and analyzed using Powerlab Chart 5 software (ADInstruments). Muscle wet weight was determined at the end of each experiment, and contraction amplitudes were normalized as grams of tension per gram of wet tissue and expressed as a percentage of baseline values, which were taken as 100%.
Evaluation of Inotropic Effect of Drugs on Mouse Diaphragm Preparation.
The time course of effects of 1 to 1000 nM clenbuterol or 1 to 1000 nM fenoterol on the amplitude of diaphragm isometric contraction was evaluated. To determine whether β2-AR agonists were able to activate Gi protein, the effect of 1 μM clenbuterol or fenoterol on diaphragm contraction was evaluated in muscle strips preincubated for 1 h with pertussis toxin (PTX) (1 μg/ml) or vehicle (Tyrode's solution containing 1 mg/ml bovine serum albumin) and compared with those of 1 μM forskolin, an direct activator of AC. Finally, the involvement of the “extracellular cAMP-adenosine pathway” on clenbuterol-induced inotropic effect was analyzed in diaphragm strips pretreated for 30 min with the organic anion transporter inhibitor probenecid (100 μM), 5-amino-9-chloro-2-(2-furyl)1,2,4-trizolo[1,5-c]quinazoline (CGS-15943) (100 nM; a nonselective adenosine receptor antagonist), and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (50 nM; a selective A1 adenosine receptor antagonist) and compared with those obtained with 10 μM cAMP, 100 μM 8-Br-cAMP, 1 μM adenosine, or 20 μM rolipram.
Drugs and Chemical Reagents.
4-[3-(Cyclopentyloxy)-4-methoxyphenyl] pyrrolidin-2-one (rolipram) was purchased from Tocris Bioscience (Ellisville, MO). Adenosine (ADO), cAMP, CGS-15943, 8-Br-cAMP, clenbuterol hydrochloride, dimethyl sulfoxide, DPCPX, fenoterol hydrobromide, forskolin, α,β-methyleneadenosine 5′-diphosphate sodium salt (AMPCP), pertussis toxin (PTX), probenecid, d-tubocurarine hydrochloride, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Data are presented as mean ± S.E.M. Statistical significance was tested by Student's t test or one-way ANOVA with a Newman-Keuls multiple comparison post hoc test using GraphPad Prism software for Windows (version 5.01; GraphPad Software Inc., San Diego, CA). Differences were considered significant at P < 0.05.
β2-Adrenoceptor Agonists Induce Transient Positive Inotropic Effect on Skeletal Muscle Contraction Force.
Figure 1, A and B, shows the time course of 1 to 1000 nM clenbuterol effects on contraction amplitude of mouse diaphragm. The β2-AR agonist induced a concentration-dependent increase in contractile force that peaked 30 min after stimulation onset. Of interest, although 1 to 100 nM clenbuterol was able to progressively increase diaphragm contraction force by up to 43% of basal value (Fig. 1, A and C), the maximal positive inotropic effect was attenuated at concentrations higher than 100 nM, reaching 123% of basal twitch contraction (100%) at 1 μM (Fig. 1, B and C).
The transient inotropic effect of clenbuterol was mimicked by the short-acting β2-AR agonists fenoterol (Fig. 2), which increased by up to 32% the diaphragm contractile force at 30 nM. The maximum inotropic effect of fenoterol was obtained ∼35 to 40 min after stimulation onset and, as observed with clenbuterol (Fig. 1, B and C), concentrations higher than 30 nM were less efficacious in bioactivity (Fig. 2, B and C).
Attenuation of Positive Inotropic Effect Induced by β2-Adrenoceptor Agonists Involves Activation of Pertussis Toxin-Sensitive Gi Protein.
To further investigate whether attenuation of the maximum inotropic response observed at high concentrations of agonists was due to coupling of β2-AR to Gi protein, the effects of clenbuterol and fenoterol on skeletal muscle contraction were evaluated in diaphragm strips preincubated for 1 h with PTX (1.0 μg/ ml). As shown in Fig. 3A, PTX potentiated the inotropic effect of 1 μM clenbuterol, restoring it to the maximum potentiation seen at 100 nM (43%) (Fig. 1B).
PTX also potentiated the inotropic effect of 1 μM fenoterol (Fig. 3B), which reached the maximum response seen at 30 nM (Fig. 2B). Moreover, PTX significantly attenuated the late descending phase of the inotropic effect induced by a high concentration of clenbuterol or fenoterol (Fig. 3, A and B), indicating that a PTX-sensitive Gi protein mediates the delayed attenuation of the β2-AR inotropic effect.
Of interest, the transient inotropic effect triggered by β2-AR agonists was mimicked by direct activation of AC with forskolin (Fig. 3C). Furthermore, as observed under β2-AR stimulation, pretreatment of diaphragm with PTX also abolished the descending phase of the forskolin inotropic effect, indicating that activation of PTX-sensitive Gi protein by β2-AR in diaphragm is triggered by signaling events downstream of AC activation. The selective inhibitor of cAMP phosphodiesterase 4, rolipram, also induced a transient positive inotropic effect in diaphragm preparation (Fig. 4A), supporting the involvement of cAMP in both ascending and descending phases of the β2-AR-mediated-positive inotropic response. In addition, considering that rolipram selectively inhibits degradation of cAMP, with no effect on other cyclic nucleotides such as cGMP, cCMP, or cUMP (Reinecke et al., 2011), this result, associated with those of forskolin, demonstrates that a Gi-related effect of β2-AR agonists depends exclusively on the cAMP signaling pathway.
β2-Adrenoceptor Coupling to Gi Protein Involves Extracellular cAMP-Adenosine Pathway and Activation of Adenosine Receptors.
Taking into account the fact that β2-AR-dependent increase of intracellular cAMP is followed by efflux of cyclic nucleotide to interstitial space (Chiavegatti et al., 2008), we assessed the possible implication of the extracellular cAMP-adenosine pathway on the biphasic pattern of inotropism induced by β2-AR agonists by analyzing the effect of cAMP, adenosine, and 8-Br-cAMP on muscle contraction.
Surprisingly, although cAMP induced a negative inotropic effect, its cell-permeable and PDE-resistant analog 8-Br-cAMP induced a sustained positive inotropic effect that persisted for at least 2 h (Fig. 4B), indicating that the final inotropic effect depends on the site of cyclic nucleotide action: the extracellular or the intracellular compartment.
As observed with cAMP, incubation of diaphragm preparation with adenosine also resulted in a negative inotropic effect (Fig. 5A). The delayed onset of cAMP and adenosine action overlapped with the descending phase of forskolin-induced (Fig. 5A) and β2-AR-induced inotropic effects (Fig. 3, A and B), indicating that extracellular cAMP and its metabolite adenosine reproduce the activation of Gi protein induced by β2-AR agonists. In fact, preincubation of diaphragm strips with PTX abolished the negative inotropic effect of cAMP (Fig. 5B), showing that extracellular cAMP is able to activate a Gi protein signaling pathway. Of more importance, the negative inotropic effect of cAMP (Fig. 5C) and the descending phase of the clenbuterol-induced inotropic effect (Fig. 6A) were inhibited by preincubation of diaphragm with the nonselective adenosine receptor antagonist CGS-15943. Furthermore, the A1 receptor antagonist DPCPX completely inhibited the negative inotropic effect of either clenbuterol (Fig. 6B) or adenosine (Fig. 6C), demonstrating that β2-AR coupling to Gi is indirect, via activation of A1 adenosine receptors.
Finally, the connection between β2-AR stimulation and the efflux of cAMP and its extracellular degradation into adenosine is shown in Fig. 7. Preincubation of diaphragm with the organic anion transporter inhibitor probenecid (Fig. 7A), in a concentration that inhibits by 75% the efflux of cAMP (Chiavegatti et al., 2008), or with the ecto-5′-nucleotidase inhibitor AMPCP (Fig. 7B) prevented the descending phase of the positive inotropic effect elicited by clenbuterol. AMPCP also abolished the cAMP-negative inotropic effect. Taken together, these results show that β2-AR coupling to Gi depends on the efflux of cAMP via a probenecid-sensitive transporter and extracellular generation of adenosine, which is schematically illustrated in Fig. 8.
The present study provides strong evidence for a dual coupling of β2-AR to Gs and Gi protein at skeletal muscle, with a significant impact on muscle inotropic state. Of more importance, the secondary coupling of β2-AR to Gi protein seems to be indirect and dependent on extracellular degradation of cAMP into adenosine, which in turn activates muscle A1 adenosine receptors.
By studying the time course of clenbuterol and fenoterol effects on diaphragm twitch contraction, here we show that the well established β2-AR positive inotropism (Bowman and Nott, 1969) is attenuated after prolonged exposure (>30 min) to agonists (Figs. 1 and 2). At high concentrations of β2-AR agonists, the decline phase of inotropic effect was so intense that it abolished the positive inotropism.
The ability of PTX to attenuate the descending phase of β2-AR positive inotropism (Fig. 3) supports the idea of dual coupling of β-AR to Gs and Gi proteins, which seems to depend on the concentration and/or exposure time to agonists. At low concentrations, β2-AR agonists induce a more sustained increase in twitch contraction (Figs. 1A and 2A), supporting the preferential coupling of β2-AR to Gs protein in skeletal muscle (Andrade-Lopes et al., 2011). On the other hand, secondary coupling of β2-AR to Gi protein would be accountable for either the descending phase of the β2-AR-positive inotropic response, observed after persistent (≥30 min) activation of β2-AR with low to intermediary concentrations of agonists or reduced inotropic response at a high concentration of clenbuterol or fenoterol (≥100 nM). The coupling of β2-AR to Gi can be used to explain previous puzzling results obtained by McCormick et al. (2010), showing that extremely high concentrations of clenbuterol (5–150 μM) reduced the contraction force of mouse extensor digitorum longus and soleus muscles.
Dual coupling of β2-AR to Gs and Gi proteins has been reported in several cells and tissues including cardiac myocytes from different species (Communal et al., 1999; Xiao et al., 1999; Hasseldine et al., 2003), mouse pulmonary artery (Wenzel et al., 2009), cultured endothelial cells, and rat carotid artery (Ciccarelli et al., 2007). However, the precise mechanism underlying the switch of β2-AR from Gs to Gi signaling remained obscure because it was primarily based on disruption of Gi signaling by PTX or immunoprecipitation of GTP-Gi protein complexes induced by β2-AR agonists (Daaka et al., 1997; Ciccarelli et al., 2007; Liu et al., 2009; Wenzel et al., 2009; Wang et al., 2011).
A plausible explanation for reduced coupling of β2-AR to Gs is receptor desensitization and reduced receptor efficacy in stimulating AC (Bünemann et al., 1999). This phenomenon depends on sequential phosphorylation of the agonist-activated receptor by specific GPCR kinases (Ferguson et al., 1998) and interaction of phosphorylated receptor with β-arrestin, which results in dissociation of receptor from G protein (for review, see Whalen et al., 2011). However, in the present study, the contribution of GPCR kinase-dependent receptor desensitization on the descending phase of β2-AR inotropism was discarded on the basis of the ability of forskolin (a direct AC activator that bypasses β2-AR) (Fig. 3C) and rolipram (a selective PDE4 inhibitor) (Fig. 4A) to induce similar biphasic inotropic effects. In fact, by showing that the descending phase of the forskolin inotropic effect is also inhibited by PTX (Fig. 4A), our data indicate that β2-AR/Gi coupling is indirect and dependent on signaling events downstream of AC activation.
Therefore, taking into account previous studies from our group showing that β2-AR agonists and forskolin are able to sequentially increase cAMP efflux from skeletal muscle cells and the extracellular adenosine level (Godinho and Costa, 2003; Chiavegatti et al., 2008), we evaluated the possible contribution of the extracellular cAMP-adenosine pathway to the biphasic inotropic effect of β-AR agonists. In support of the above hypothesis, incubation of diaphragm with cAMP resulted in a PTX-sensitive negative inotropic effect (Fig. 5B). The time course of cAMP effect remarkably resembled the late attenuation phase of β2-AR positive inotropism. In fact, the delayed descending phase of the clenbuterol/fenoterol/forskolin inotropic effect presented here coincides with the increment of extracellular cAMP induced by the β-AR agonist isoproterenol or forskolin in cultured skeletal muscle or in rat skeletal muscle (Godinho and Costa, 2003; Chiavegatti et al., 2008). Furthermore, the Gi-dependent effects of clenbuterol and cAMP were mimicked by adenosine and inhibited by adenosine receptor antagonists CGS-15943 (nonselective) and DPCPX (A1-selective) (Figs. 5C and 6, A–C), indicating that the cAMP effect depends on activation of adenosine receptors. Of more importance, selective inhibition of the descending phase of clenbuterol inotropic effect by either probenecid or the ecto-5′-nucleotidase inhibitor AMPCP (Fig. 7, A and B) definitively supports the indirect coupling of β2-AR to Gi, via extracellular cAMP-adenosine pathway, which is illustrated in Fig. 8.
In view of the expression of distinct adenosine receptors subtypes (A1, A2A-2B, and A3) coupled to Gi/Gs proteins in skeletal muscle (Ren and Stiles, 1994; Lynge and Hellsten, 2000; Reading and Barclay, 2001; Thong et al., 2007), the interstitial formation of adenosine via ecto-5′-PDE and ecto-5′-nucleotidase (Hellsten and Frandsen, 1997; Chiavegatti et al., 2008) may allow the autocrine regulation of skeletal muscle function. This is of special clinical importance because β2-AR agonists, used for treatment of asthma or chronic obstructive pulmonary disease, transiently improve isometric and isotonic contractility of respiratory skeletal muscle, including the diaphragm (Van Der Heijden et al., 1998), which is compromised in patients with chronic obstructive pulmonary disease (Ottenheijm et al., 2007). In addition, under pathological conditions, increased β2-AR-Gi coupling could result in impaired contraction function, as observed in muscle-specific Gs-deficient mice (Chen et al., 2009).
Although the cross-talk between β2-AR and adenosine receptors has been described in several cell types, the involvement of the extracellular cAMP-adenosine pathway on β2-adrenergic signaling has been overlooked. This new level of GPCR cross-talk may explain the inhibitory effect of adenosine on isoproterenol-induced activation of AC in rat cardiac membranes (LaMonica et al., 1985), adipocytes (Elks et al., 1987), and smooth muscle (Gerwins et al., 1990) cells lines. In addition, the extracellular cAMP-adenosine pathway would elucidate why in rat perfused heart, catecholamines increase extracellular adenosine formation, which in turn prevents full mechanical responsiveness to β-AR stimulation (Dobson et al., 1986). In stress conditions, such as hypoxia or ischemia, increased extracellular adenosine levels are responsible for cardioprotective effects, which, at least in part, involve activation of Gi-coupled adenosine A1 and A3 adenosine receptors (Safran et al., 2001; Du et al., 2012).
As observed in heart, β-AR improves skeletal muscle twitch contraction by increasing intracellular Ca2+ availability (Lynch and Ryall, 2008). However, long-lasting elevation of cytosolic Ca2+ may have deleterious consequences, involving activation of the Ca2+-dependent proteases (Gissel, 2005; Verburg et al., 2009). Again, extracellular adenosine exerts a protective effect in skeletal muscle, mainly via activation of A1 and A3 subtype receptors (Zheng et al., 2007). Thus, the cross-talk between Gs-coupled β2-AR and Gi-coupled adenosine receptors presented here possibly provides a feedback mechanism for fine control of intracellular cAMP levels, which results in attenuation of β responsiveness and modulation of skeletal muscle contraction.
In summary, this study adds a new view to the actual concept of promiscuous receptor/G protein coupling by showing the indirect coupling of β2-AR to Gi protein, which depends on the activation of adenosine receptors. In skeletal muscle, this extracellular arm of cAMP signaling illustrated in Fig. 8 provides a negative feedback loop, which may limit stimulatory G protein-coupled receptor response and possible harmful exacerbation of muscle contraction. In fact, considering the increased generation of extracellular adenosine during muscle contraction (Lynge et al., 2001), the extracellular cAMP-adenosine pathway may influence many other aspects of muscle physiology via activation of postsynaptic adenosine receptors, including those associated with the regulation of skeletal muscle carbohydrate metabolism (Hespel and Richter, 1998) and muscle proteolysis (Gissel, 2005; Bergantin et al., 2011). Thus, in skeletal muscle, the extracellular cAMP-adenosine pathway may function as a feedback mechanism able to modulate the cAMP signaling events initiated by other endogenous substances through activation of G-protein-coupled receptors, such as adrenoceptors or calcitonin gene-related peptide, are able to induce the efflux of cAMP (Godinho and Costa, 2003).
In fact, it is important to emphasize that efflux of cAMP/cGMP seems to be a widespread signaling mechanism (Hofer and Lefkimmiatis, 2007; Sager and Ravna, 2009) reported in vascular smooth muscle cells (Dubey et al., 1996), cardiac fibroblasts (Dubey et al., 2001), oviduct cells (Cometti et al., 2003), kidney (Jackson and Raghvendra, 2004; Dubey et al., 2010), adipose tissue (Strouch et al., 2005) gastrointestinal tract (Giron et al., 2008), human placenta explants (Biondi et al., 2010), astrocytes, and microglial cells (Verrier et al., 2011). Thus, the extracellular cAMP-adenosine pathway may represent a general autocrine and/or paracrine mechanism that indirectly modulates the signaling triggered by distinct receptors coupled to Gs proteins, qualifying cyclic nucleotides as extracellular third messengers, and the extracellular cAMP-adenosine signaling pathway as a potential pharmacological target for therapeutic intervention. Additional studies are necessary to determine the contribution of other extracellular cyclic nucleotides to GPCR signaling cascade.
Participated in research design: Duarte, Menezes-Rodrigues, and Godinho.
Conducted experiments: Duarte, Menezes-Rodrigues, and Godinho.
Performed data analysis: Duarte, Menezes-Rodrigues, and Godinho.
Wrote or contributed to the writing of the manuscript: Godinho.
We thank Marcelo Pires-Oliveira for helpful comments on the manuscript and Maria do Carmo Gonçalo, Alex Sandro F. Oliveira, and Celso Moreira das Dores for excellent technical assistance.
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.
- G protein-coupled receptor
- adenylyl cyclase
- multidrug resistance protein
- α,β-methyleneadenosine 5′-diphosphate sodium salt
- pertussis toxin
- analysis of variance.
- Received February 8, 2012.
- Accepted March 20, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics