protective action of anti-Gbc treatment in preventing induction of the airway asthmatic response, and it remains to be determined whether this treatment is also effective in reversing the asthmatic response once manifested. In further considering the present observations, it should be noted that, like pretreatment with the anti-Gbc blocking peptide, the induction of in vivo AHR and altered agonist responsiveness in the isolated sensitized ASM tissues were also abrogated by pretreatment with gallein, a small molecule inhibitor of Gbc signaling [17,26,27]. Unlike the anti-Gbc blocking peptide, however, gallein did not suppress the pulmonary inflammatory response in OVA-sensitized+challenged rabbits (Fig. 3). While the disparity between these inhibitors with respect to their anti- inflammatory actions is not readily explained, one possibility relates to potential differences in their pharmacodynamic or pharmacokinetic properties that might influence their effects on the effector systems that regulate AHR and inflammation. Another reasonable explanation relates to potential differences in their mechanisms of Gbc inhibition under the present experimental conditions. In this regard, it has been proposed that, among the small molecule inhibitors of Gbc signaling examined to date, each has its distinctive spacial orientation of binding to the bioactive site (‘‘hot spot’’) on the Gbc surface, thereby accounting for inhibition of only specific Gbc-targeted effector interactions [17]. Accord- ingly, in extending this concept to the present observations, it is conceivable that gallein allows only for inhibition of those Gbc- targeted effector interactions that regulate airway responsiveness but not those that mediate allergic lung inflammation, although it should be noted that gallein was found to inhibit carrageenan- induced footpad inflammation in mice [27]. On the other hand, by sequestering the Gbc subunit, the anti-Gbc blocking peptide is arguably capable of relatively greater inhibition of Gbc interac- tions with different effector targets, including those that mediate both changes in airway responsiveness and inflammation. This interesting possibility remains to be systematically investigated. In evaluating the implications of the present study, it must be emphasized that our observations pertain to studies conducted in an in vivo rabbit model of allergic airway disease and in isolated sensitized rabbit ASM tissues. Therefore, the extent to which these observations relate to the human condition is open to speculation. In this respect, it is noteworthy that our results are consistent with recent evidence that implicates upregulated PDE4 activity in mediating the airway responses to allergen challenge in asthmatic individuals [28], and the induction of altered HASM function [10,11,23,56,57]. Additionally, our data generated in the rabbit ASM tissues are consistent with those obtained in the cultured HASM cells, as both these experimental preparations exhibited complementary Gbc-dependent changes in ASM function. These included compatible effects of specific inhibitors on the Gbc- regulated signaling events mediating IL-13-induced PDE4 expres- sion in HASM cells (Figs. 7 and 8) and altered responsiveness in rabbit ASM tissues (Fig. 9). Moreover, our findings regarding the Gbc-regulated mechanism underlying the effects of IL-13 on ASM responsiveness are consistent with those in previous reports demonstrating that IL-13 evokes downstream ERK1/2-dependent changes in HASM contractility [58] and intracellular calcium mobilization [59], as well as pulmonary inflammation [60]. Regarding the latter, it should be noted that our in vivo observations in rabbits,concur with those in previous reports that also demonstrated a predominant neutrophilic pulmonary inflam- matory response initially (i.e., up to 24 hours) following antigen challenge in sensitized mice [61–63] and rabbits [64], as well as the reported predominant neutrophilic inflammatory response detected in BAL samples obtained hours following airway antigen G Protein bc-Subunit Signaling in Asthma challenge in allergic asthmatic individuals [65]. In this regard, however, it is important to note that, given the frequent observance of eosinophilic infiltration at 24 hours after allergen challenge in other studies in OVA-sensitized mice, consideration must be given to other factors that may also contribute to our observed neutrophilic response, including LPS contamination of the administered OVA preparation. Finally, another relevant issue is that our observed changes in constrictor and relaxant responsiveness in the sensitized rabbit ASM tissues mimicked the perturbations in airway function that characterize the human asthmatic ASM phenotype, including enhanced constrictor responsiveness to cholinergic stimulation and impaired b2AR- mediated airway relaxation [66,67]. Thus, in view of these considerations, we believe that the findings of the present study are applicable, at least in part, to the human condition. In conclusion, this study is the first to report that Gbc signaling associated with ERK1/2 activation and upregulated PDE4 activity plays a critical role in mediating the induction of airway hyperresponsiveness and inflammation in a rabbit model of allergic asthma. By identifying that Gbc signaling mediates these key characteristic features of the airway asthmatic phenotype, the present findings support the consideration that future interventions targeted at modulating Gbc function may yield new approaches to treat allergic airway disease. Methods Materials All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise indicated. Human ASM (HASM) cells were obtained from Bio Whittaker, Inc. Animals Thirty-five young adult (6–8 months of age) male New Zealand White rabbits purchased from Covance were used in this study, which was approved by the Biosafety and Animal Research Committee of the Research Institute at Children’s Hospital of Philadelphia. OVA sensitization and challenge Rabbits were actively immunized with weekly intraperitoneal (i.p.) injections of 1 ml of an OVA-containing emulsion, comprising 2.5 mg OVA prepared from lyophilized powder (Grade V; $98% purity) in 1 part physiological saline and 1 part of Alum adjuvant (Pierce), for a total of 4 injections. One week later, similar to the approach previous described in OVA- sensitized rabbits [68], the animals received a single inhalation challenge of an aerosolized 7% solution of OVA in physiological saline, delivered at a flow rate of 8 l/min over ,20 min via a compressor nebulizer (DeVilbiss, PulmoMate) connected to an oro-pharyngeal tube. The OVA challenges were conducted in the absence and presence of pre-nebulization at 1 hr prior to antigen challenge either with 0.5 mg/Kg of the inhaled glucocorticoster- oid, budesonide (Pulmicort Respules; Astra-Zeneca), 1 mg/Kg of a cell permeable anti-Gbc blocking peptide (AnaSpec), comprised of a membrane permeable sequence (MPS; 15 amino acids) conjugated to the Gbc-sequestering C-terminal domain (28 amino acids) of phosducin-like protein [25], or 1 mg/Kg of the inert MPS peptide alone serving as a negative-control. The blocking Gbc peptide and control MPS aerosolized solutions were prepared in 3 ml of physiological saline and nebulized over 15–20 min. In separate studies, at 1 hr prior to OVA challenge, control and OVA-sensitized rabbits were pretreated with an i.p. bolus injection of either vehicle alone (PBS) or 30 mg/Kg of gallein [26], a PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e32078