G Protein bc-Subunit Signaling in Asthma Figure 1. Inhibition of ERK1/2, PDE4, or Gbc signaling prevents induced changes in agonist responsiveness in OVA-serum- sensitized ASM tissues. Relative to vehicle- or control serum-exposed rabbit ASM tissues, tissues passively sensitized for ,18 hr with OVA serum exhibit significantly increased contractility to ACh (A) and impaired relaxation to isoproterenol (B). Pre-treatment with either U0126, rolipram, or anti- Gbc peptide prevents OVA serum-induced changes in ASM responsiveness. Data are mean 6 SD values from 5–7 experiments. ANOVA used for multiple comparisons of mean Tmax values. *p,0.05; **p,0.01. doi:10.1371/journal.pone.0032078.g001 control rabbits that were treated with anti-Gbc peptide (i.e., Rrs- max = 2.8260.63-fold above baseline; n = 3); 2) the AHR evoked by antigen challenge was not significantly altered in OVA- sensitized rabbits that were treated with MPS alone (i.e., Rrs- max = 6.9560.84-fold above baseline; n = 3); and 3) suppression of AHR comparable to that observed in anti-Gbc blocking peptide- or budesonide-treated rabbits was also detected in OVA-sensitized rabbits that were pretreated with gallein (30 mg/Kg; n = 4), a small molecule inhibitor of Gbc signaling [17,26,27], yielding Rrs- max values of 2.5460.72-fold above baseline. Finally, in accordance with the above observations, comparable results were also obtained when analyzing MCh responsiveness in terms of the corresponding changes in dynamic compliance (Cdyn) measured under the different experimental conditions. The baseline Cdyn values were lower in the OVA-sensitized+challenged vs. control rabbits, averaging 3.7160.06 vs. 4.4560.04 ml/cmH20, respec- tively (p,0.05) and, as depicted in Fig. S2: 1) relative to controls, MCh-induced decreases in Cdyn (expressed as % of baseline) were significantly greater in the OVA-sensitized+challenged rabbits; and 2) this induced change in the Cdyn responses to MCh was suppressed in OVA-sensitized rabbits that were treated with anti- Gbc blocking peptide before antigen challenge, similar to the suppression exhibited in OVA-sensitized rabbits that were pretreated with budesonide. As with the above Rrs data, PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32078