G Protein bc-Subunit Signaling in Asthma Figure 9. Inhibition Gbc signaling prevents rolipram-sensitive changes in agonist responsiveness in IL-13-exposed rabbit ASM tissues. Relative to controls, ASM tissues sensitized with IL-13 (50 ng/ml624 hr) exhibit significantly increased contractility to ACh (A) and impaired relaxation to isoproterenol (B). Inhibition of Gbc signaling by pretreatment with anti-Gbc blocking peptide or gallein, or inhibition of PDE4 activity with rolipram, prevents IL-13-induced changes in ASM responsiveness. Data are mean 6 SD values from 5–6 experiments. ANOVA used for multiple comparisons of mean Tmax and Rmax values. *p,0.05; **p,0.01. doi:10.1371/journal.pone.0032078.g009 in concert with the latter finding, we previously demonstrated that the anti-Gbc blocking peptide abrogates PTX-sensitive ERK1/2 activation and its consequent induction of PDE4-mediated changes in responsiveness in b2AR-desensitized ASM, whereas comparable treatment with a MPS-conjugated anti-Gia3 peptide has no effect [11]. Consistent with this evidence, our present observations demonstrated that, together with its protective effects in OVA serum- and IL-13-sensitized isolated ASM tissues (Figs. 1 and 9, respectively), pretreatment with aerosolized anti-Gbc blocking peptide prevented in vivo AHR evoked by antigen challenge in OVA-sensitized rabbits (Fig. 2). Moreover, pretreat- ment with anti-Gbc blocking peptide also suppressed the pulmonary inflammatory response and reversed the predominance of IL-13 vs. IFN-c cytokine levels in the lungs of the OVA- sensitized+challenged rabbits (Figs. 3 and 5). Of significance, these observations, as well as those identifying that the Gbc signaling mechanism activated in the sensitized ASM involves ERK1/2- dependent upregulation of PDE4 activity, concur with evidence provided in earlier in vivo studies demonstrating that: 1) inhibition of ERK1/2 activation suppresses both the in vivo AHR and pulmonary inflammation elicited by antigen challenge in OVA- sensitized mice [54,55]; and 2) increased PDE4 activity plays a decisive role in mediating both the heightened bronchoconstrictor responsiveness and airway inflammation evoked by allergen challenge in asthmatic subjects [28] and in animal models of allergic asthma [29–33]. Thus, when considered in light of the extended mechanistic observations described herein, the circum- stantial evidence provided by the latter findings further substan- tiate the concept that Gbc signaling associated with ERK1/2- dependent induction of PDE4 activity is importantly involved in mediating both the altered airway responsiveness and inflamma- tion exhibited in the allergic asthmatic lung. In this context, it is important to indicate that the critical role identified herein for Gbc signaling is based on evidence demonstrating the broncho- PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e32078