of this evidence, together with that implicating an important causal relationship between PTX-sensitive Gbc signaling and inflamma- tion [17–22], the present study addressed the hypothesis that Gbc signaling regulates the altered airway responsiveness and inflam- mation exhibited in the allergic asthmatic state. The results obtained in studies conducted in an in vivo rabbit model of allergic asthma and in isolated atopic sensitized ASM tissues are the first to demonstrate that: 1) inhibition of Gbc signaling prevents the induction of airway hyperresponsiveness and inflammation elicited by antigen challenge in allergic rabbits, as well as the pro- asthmatic changes in constrictor and relaxation responsiveness exhibited in atopic sensitized ASM tissues; and 2) these bronchoprotective actions of Gbc inhibition are attributed to suppression of Gbc-induced direct activation of c-Src, which leads to downstream ERK1/2-dependent upregulation of PDE4 activity and its consequent pro-asthmatic effects on airway function. Taken together, these new findings highlight a heretofore- unidentified pivotal role for Gbc signaling in regulating the airway asthmatic phenotype, and suggest that interventions targeted at suppressing Gbc signaling associated with Gi protein activation may lead to new approaches to treat allergic airway disease. Results Gbc-coupled ERK1/2 and PDE4 activation mediates altered constrictor and relaxation responsiveness in atopic sensitized ASM Given recent evidence demonstrating that transcriptional upregulation of PDE4 activity due to Gi-bc-regulated activation of ERK1/2 mediates proasthmatic changes in agonist responsive- ness in b2AR-desensitized ASM [10,11,23], we initially examined whether these signaling molecules also participate in mediating the reported IgE-induced Gi protein-dependent proasthmatic changes in responsiveness exhibited in ASM tissues passively sensitized with atopic asthmatic serum [7,24]. Accordingly, agonist-induced constrictor and relaxation responses were compared in isolated na¨ıve rabbit ASM tissues that were incubated overnight with vehicle alone (control) or serum isolated from either non-sensitized (control serum) or OVA-sensitized rabbits at 24 hr following OVA inhalation (OVA serum), both in the absence and presence of pretreatment with either the PDE4 inhibitor, rolipram (10 mM), the ERK1/2 inhibitor, U0126 (5 mM), or a Gbc sequestering (blocking) peptide (20 mM) comprised of the C-terminal domain of phosducin-like protein (PhLP) conjugated to an inert membrane permeable peptide (MPS) carrier [25]. The latter anti-Gbc blocking peptide was previously shown to inhibit Gi protein- dependent upregulation of PDE4 activity and its consequent induction of altered responsiveness in b2AR-desensitized ASM [11]. As shown in Fig. 1A, relative to the similar responses obtained in vehicle- and control serum-exposed tissues, OVA serum-sensitized ASM tissues exhibited significantly increased constrictor responsiveness to cumulative administration of ACh, yielding a mean 6 SD maximal constrictor response (Tmax) that averaged 121.7616.7% of control (p,0.05). This enhanced contractility was completely abrogated in OVA serum-sensitized tissues that were pretreated either with the anti-Gbc blocking peptide, rolipram, or U0126, with no significant differences observed between the protective effects of these inhibitors. Under the same treatment conditions, cumulative administra- tion of the b2AR agonist, isoproterenol, produced dose-dependent relaxation of half-maximally pre-constricted ASM segments (Fig. 1B). Relative to controls, the relaxation responses were significantly attenuated in the OVA serum-sensitized tissues, G Protein bc-Subunit Signaling in Asthma wherein the mean 6 SE maximal relaxation response (Rmax) amounted to 27.8612.1% vs. 46.1611.1% in the controls (p,0.01). This impaired relaxant responsiveness was also ablated in OVA serum-exposed tissues that were pretreated either with the anti-Gbc blocking peptide, rolipram, or U0126, with no significant differences detected between the protective effects of these inhibitors. Of note, results obtained in related experiments demonstrated that: 1) relative to untreated (vehicle-exposed) tissues, neither the Tmax nor Rmax responses to ACh and isoproterenol, respectively, were significantly affected in control serum-exposed ASM tissues that were pretreated either with the inert MPS carrier peptide alone or anti-Gbc blocking peptide; and 2) in contrast to the protective effects of the anti-Gbc peptide in OVA serum-sensitized ASM tissues, pretreatment of these tissues with MPS alone did not significantly affect their altered agonist responsiveness (Fig. S1). Moreover, as we previously reported in na¨ıve rabbit ASM tissues [10,11,23], neither co-incubation with rolipram, U0126, nor the anti-Gbc peptide significantly affected the responsiveness of control serum-exposed ASM tissues to either ACh or isoproterenol (data not shown). Thus, in demonstrating that inhibition of either Gbc, ERK1/2 or PDE4 signaling prevents the changes in agonist responsiveness elicited in OVA serum- sensitized ASM, these observations are consistent with those previously reported in b2AR-desensitized ASM wherein activation of the Gbc subunit of Gi protein was also found to initiate ERK1/ 2-dependent rolipram-sensitive proasthmatic changes in constric- tor and relaxation responsiveness [10,11,23]. Gbc signaling mediates in vivo airway hyperresponsiveness in allergic rabbits To determine whether Gbc inhibition exerts a comparable bronchoprotective action in vivo, we examined the effect of pretreatment with aerosolized anti-Gbc blocking peptide on bronchoconstrictor responsiveness to MCh in OVA-sensitized rabbits, and compared this effect to that of the inhaled glucocorticosteroid, budesonide. The measurements of baseline respiratory system resistance (Rrs) obtained at 24 hr following antigen challenge in non-sensitized (control) and OVA-sensitized rabbits were not significantly different, averaging 0.02960.004 and 0.03160.005 cmH20/ml/sec, respectively. Relative to controls, however, the OVA-sensitized rabbits exhibited pro- nounced AHR to i.v. administration of MCh, as evidenced by markedly heightened dose-dependent increases in Rrs (Fig. 2). The mean 6 SE maximal MCh-induced increase in Rrs (Rrs-max) averaged 7.8160.74-fold above baseline in the OVA-sensitized rabbits vs. an increase of 3.0160.60-fold in the control animals (p,0.001). This AHR was virtually completely suppressed in OVA-sensitized rabbits that were treated with inhaled anti-Gbc blocking peptide prior to OVA challenge, similar to the suppression of AHR exhibited in OVA-sensitized rabbits that were pretreated with inhaled budesonide (Fig. 2). Of note, comparable significant differences were also detected when analyzing these data using the nonparametric Kruskal-Wallis test (p = 0.023), with the Dunn’s post-test demonstrating a significant difference (p,0.05) when comparing the median Rrs-max value in the control (2.69; range: 2.10–4.89) vs. non-pretreated OVA sensitized rabbits (7.29; range: 6.05–9.84), whereas no significant difference was detected when comparing the median of the controls vs. median Rrs-max of 3.22 (range: 6.95–9.84) obtained in the OVA-sensitized animals that were pretreated with the anti- Gbc blocking peptide. In relation to these observations, it should be noted that results generated in separate experiments demon- strated that, relative to the above corresponding determinations: 1) bronchoconstrictor responsiveness to MCh was unaffected in PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32078