Figure 4. Pulmonary inflammatory response to antigen challenge in OVA-sensitized rabbits. Representative high magni- fication photomicrographs (mag. 61000) demonstrating that inflam- matory infiltrate in lung tissues (A) and BALF (B) isolated from OVA- sensitized+challenged rabbits is mostly composed of neutrophils (N) with lesser amounts of mononuclear macrophages (M) and rare eosinophils (E). doi:10.1371/journal.pone.0032078.g004 which encodes the C-terminal domain of bARK1 that was shown to block Gbc signaling [14,15], both at a multiplicity of infection (MOI) of 100. Whereas cells transfected with adeno-LacZ exhibited IL-13-induced c-SrcTyr416 and ERK1/2 phosphoryla- tion, this effect of IL-13 was prevented in cells transfected with adeno-bARK-ct (Fig. 7B). In light of the above results implicating Gbc as an upstream regulator of IL-13-induced c-Src activation, co-immunoprecipita- tion studies were then conducted to determine whether IL-13 evokes a direct interaction between activated Gbc and phosphor- ylated c-Src. The immunoblots in Fig. 7C demonstrate that, relative to unstimulated cells, enhanced co-localization of phosphorylated c-SrcTyr416 with immunoprecipitated Gb was detected in IL-13-treated HASM cells (Fig. 7C; top panel), and that this induced association was abrogated in IL-13-exposed HASM cells that were pretreated with either anti-Gbc blocking peptide or gallein, as well as in IL-13-exposed cells that were transfected with adeno-bARK-ct. Of note, these results were obtained under similar conditions of loading of immunoprecipi- tated Gb, as evidenced by the immunoblots using anti-Gb antibody in Fig. 7C (bottom panel). Thus, together with the above observations, these data support the concept that IL-13 stimulates direct coupling of activated Gbc to c-Src, and that this signaling event is associated with ERK1/2 activation in the cytokine-exposed ASM. G Protein bc-Subunit Signaling in Asthma Gbc regulates IL-13-induced transcriptional upregulation of PDE4: Role in mediating altered ASM responsiveness To further elucidate the mechanism by which Gbc regulates the induction of PDE4 activity, we next investigated the effects of inhibition of Gbc signaling and its suspected downstream effectors on IL-13-induced PDE4 expression in HASM cells. As shown in Fig. 8A, treatment of HASM cells with IL-13 (50 ng/ml624 hr) evoked temporal increases in mRNA expression of PDE4D, the functionally dominant PDE4 subclass in HASM cells [38,39], with peak expression detected at 12 hr and elevated levels sustained at 24 hr. As demonstrated by a representative experiment in Fig. 8B, and the corresponding results based on densitometric analysis of the data obtained in 4 such experiments in Fig. 8C, relative to control (vehicle-exposed) HASM cells (lane 1), IL-13-induced upregulation of PDE4D transcripts at 12 hr averaged ,7.6-fold (lane 2), and this response was suppressed by pretreating IL-13- exposed cells with previously reported [10,11,23] maximally effective concentrations of either PTX (100 ng/ml; lane 3), which ADP ribosylates Gi protein, the anti-Gbc blocking peptide (20 mM; lane 4), or the ERK1/2 inhibitor, U0126 (5 mM; lane 5). By comparison, induction of PDE4D transcripts by IL-13 was unaltered in cells pretreated with either the p38 MAPK inhibitor, SB202190 (10 mM; lane 6) or the JNK inhibitor, SP600125 (10 mM; lane 7), whereas the induction of PDE4D5 transcripts was prevented in IL-13-exposed cells that were pretreated with either the c-Src family tyrosine kinase inhibitor, SU6656 (10 mM; lane 8) or gallein (10 mM, lane 9). Thus, consistent with earlier evidence demonstrating that activation of the Gbc subunit of Gi protein elicits c-Src-induced downstream signaling via the Ras/c-Raf1/ MEK-ERK1/2 pathway [14,15], the above results support the notion that IL-13-induced PDE4 expression is regulated by Gi-bc signaling coupled to c-Src activation that, in turn, leads to downstream ERK1/2-dependent induction of PDE4D transcripts. In concert with the above results, extended studies conducted in isolated rabbit ASM tissues demonstrated that (Fig. 9): 1) as previously described [34], relative to controls, ASM tissues exposed to IL-13 (50 ng/ml624 hr) exhibit significantly height- ened constrictor responses to ACh and impaired relaxation responses to isoproterenol; 2) as in atopic serum-sensitized tissues (Fig. 1), these IL-13-induced changes in ASM responsiveness are also prevented by pretreating the tissues with either rolipram or the anti-Gbc blocking peptide; and 3) comparable inhibition of the pro-asthmatic effects of IL-13 is also seen in tissues pretreated with gallein (10 mM). Along with the above results, these observations support the concept that activation of the Gbc subunit of Gi protein, which elicits c-Src-induced downstream signaling via the Ras/c-Raf1/MEK-ERK1/2 pathway [14,15], leads to PDE4 upregulation and its consequent induction of proasthmatic changes in constrictor and relaxation responsiveness in OVA serum- and IL-13-sensitized ASM. Discussion Whereas activation of the a subunits of the G proteins, Gq and Gs, is identified as primarily responsible for mediating acute ASM contraction and relaxation, respectively, in response to various bronchoactive agents [1,40], signaling initiated by PTX-sensitive Gi proteins was shown to play a critical role in mediating the heightened constrictor and impaired relaxation responses that typify ASM tissues sensitized under different proasthmatic condi- tions [7–11]. The latter studies presumed that the proasthmatic role of PTX-sensitive Gi protein activation was attributed to an observed increase in ASM expression of the Gai2 and Gai3 isoforms [7–9]; however, it was subsequently demonstrated that transgenic mice PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32078