G Protein bc-Subunit Signaling in Asthma Figure 5. Proinflammatory cellular and cytokine responses in antigen-challenged OVA-sensitized rabbits are suppressed by anti- Gbc blocking peptide. Relative to controls, total inflammatory cell count (A) and neutrophil/macrophage cellular ratio (B), are significantly increased in BALF samples from antigen-challenged OVA-sensitized rabbits. Correspondingly, BALF levels of INF-c are significantly reduced whereas IL-13 levels are increased in OVA-sensitized+challenged animals. These proinflammatory indices are suppressed in BALF from sensitized rabbits that are treated with inhaled anti-Gbc peptide prior to OVA challenge. Data are represented by bar plots depicting median and range values. Statistical comparisons are made using the Kruskal-Wallis test. *p,0.05 based on Dunn’s post-test. doi:10.1371/journal.pone.0032078.g005 overexpressing Gai2 or a peptide inhibitor of Gai2 in ASM do not exhibit increased ASM contractility to MCh [41]. Instead, it is now generally recognized that most physiological processes inhibited by PTX are mediated by the bc subunits of Gi protein, rather than the a subunit, and that most Gbc-dependent signaling arises from Gi protein [17,42–45]. Consistent with this concept, our present observations demonstrated that inhibition of Gbc activation prevented ERK1/2-dependent rolipram-sensitive changes in con- strictor and relaxation responsiveness in rabbit ASM tissues sensitized with OVA serum or IL-13 (Figs. 1 and 9, respectively), and that this effect of Gbc inhibition was associated with suppression of the upregulated PDE4 activity detected in these sensitized tissues (Fig. 6). Moreover, inhibition of Gbc, resulting in its impaired direct activation of c-Src and accompanying ERK1/2 activation (Fig. 7), was also found to prevent PTX-sensitive induction of PDE4D mRNA transcripts by IL-13 in HASM cells (Fig. 8). Finally, in concert with the well documented causal relationship between PTX-sensitive Gbc signaling and inflamma- tion [17–22], the present results demonstrated that, together with abrogating in vivo airway hyperreactivity, inhibition of Gbc signaling also suppressed the pulmonary inflammatory response to antigen challenge in OVA-sensitized rabbits (Figs. 2 and 3, respectively), and that these protective effects of Gbc inhibition were associated with suppression of the upregulated PDE4 activity detected in the lungs of the OVA-challenged allergic rabbits (Fig. 6A). To our knowledge, these findings are the first to identify that Gbc signaling leading to c-Src-induced ERK1/2-dependent upregulation of PDE4 activity plays a decisive role in regulating the altered airway function associated with the allergic asthmatic phenotype. The present observations raise certain relevant considerations regarding potential mechanisms by which Gbc signaling regulates the proasthmatic state. In this regard, our results generally agree with the substantial body of literature reporting that PTX-sensitive Gbc signaling initiates critical proinflammatory events in various cell types [17–22], including those implicated in the pathobiology of allergic asthma. Accordingly, Gbc-induced activation of the signaling molecules, PI3K and PLCb2/b3, was shown to be an essential mechanism that regulates IgE-mediated degranulation by stimulated mast cells [18,44,45], as well as the recruitment and migration of leukocytes [17,27,46]. Moreover, it is noteworthy that PI3K and PLCb2/b3 are also key regulators of ASM proliferation and contractility, respectively [1,6,40], and that the latter regulatory action may account, at least in part, for the reported PTX-sensitive increase in ASM contractility associated with heightened PLC-mediated inositol phosholipid signaling in ASM isolated from allergic rabbits [47], as well as in rabbit ASM passively sensitized with atopic asthmatic serum [5,48]. Another mechanism relates to Gi-bc-initiated activation of MAPK signaling, particularly that attributed to stimulation by Gi-bc of c-Src-induced signaling via the Ras/Raf/ERK1/2 pathway [13– 15]. The present observations provide several lines of evidence that implicate the latter Gi-bc-regulated mechanism in mediating our observed proasthmatic changes in ASM contractility, as given by the results demonstrating key intermediate regulatory roles for Gi-bc, c-Src and ERK1/2 activation in mediating the upregulated PDE4 activity associated with altered agonist responsiveness in the sensitized ASM tissues, and induction of PDE4D transcripts by IL- 13 in HASM cells. Furthermore, our extended observations PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32078