BACKGROUND AND PURPOSEWhile selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca 2+ signalling and Ca 2+ sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca 2+ signalling and sensitivity. EXPERIMENTAL APPROACHAirways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca 2+ signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca 2+ -sensitive indicator (with or without caged-IP3). Effects on Ca 2+ sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca 2+ . KEY RESULTSThe TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca 2+ oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca 2+ signals in ASM cells. Ca 2+ increases mediated by the photolysis of caged-IP3 were also attenuated by chloroquine, quinine and denotonium. In Ca 2+ -permeabilized ASM cells, the TAS2R10 agonists dilated MCh-and 5HT-constricted airways. CONCLUSIONS AND IMPLICATIONS TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca2+ oscillations while simultaneously reducing the Ca 2+ sensitivity of ASM cells. Reduction of Ca 2+ oscillations may be due to inhibition of Ca 2+ release through IP3 receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions. [Ca 2+ ]i, cytosolic calcium concentration; ASM, airway smooth muscle; BKCa, Ca 2+ -dependent, large-conductance potassium channels; COPD, chronic obstructive pulmonary disease; IP3, inositol-1,4,5-trisphosphate; MCh, methacholine; MLC, myosin light chain; MLCK, myosin light chain kinase; PBST, 0.1% triton X-100 PBS solution; RyR, ryanodine receptor; SOC, store-operated calcium; SR, sarcoplasmic reticulum; TAS2R, taste receptor type 2 Abbreviations

The inositol trisphosphate receptor () is one of the most important cellular components responsible for oscillations in the cytoplasmic calcium concentration. Over the past decade, two major questions about the have arisen. Firstly, how best should the be modeled? In other words, what fundamental properties of the allow it to perform its function, and what are their quantitative properties? Secondly, although calcium oscillations are caused by the stochastic opening and closing of small numbers of , is it possible for a deterministic model to be a reliable predictor of calcium behavior? Here, we answer these two questions, using airway smooth muscle cells (ASMC) as a specific example. Firstly, we show that periodic calcium waves in ASMC, as well as the statistics of calcium puffs in other cell types, can be quantitatively reproduced by a two-state model of the , and thus the behavior of the is essentially determined by its modal structure. The structure within each mode is irrelevant for function. Secondly, we show that, although calcium waves in ASMC are generated by a stochastic mechanism, stochasticity is not essential for a qualitative prediction of how oscillation frequency depends on model parameters, and thus deterministic models demonstrate the same level of predictive capability as do stochastic models. We conclude that, firstly, calcium dynamics can be accurately modeled using simplified models, and, secondly, to obtain qualitative predictions of how oscillation frequency depends on parameters it is sufficient to use a deterministic model.

BackgroundTransforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.MethodsSerum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.ResultsTGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.ConclusionsRGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.

Intracellular dynamics of airway smooth muscle cells (ASMC) mediate ASMC contraction and proliferation, and thus play a key role in airway hyper-responsiveness (AHR) and remodelling in asthma. We evaluate the importance of store-operated entry (SOCE) in these dynamics by constructing a mathematical model of ASMC signaling based on experimental data from lung slices. The model confirms that SOCE is elicited upon sufficient depletion of the sarcoplasmic reticulum (SR), while receptor-operated entry (ROCE) is inhibited in such conditions. It also shows that SOCE can sustain agonist-induced oscillations in the absence of other influx. SOCE up-regulation may thus contribute to AHR by increasing the oscillation frequency that in turn regulates ASMC contraction. The model also provides an explanation for the failure of the SERCA pump blocker CPA to clamp the cytosolic of ASMC in lung slices, by showing that CPA is unable to maintain the SR empty of . This prediction is confirmed by experimental data from mouse lung slices, and strongly suggests that CPA only partially inhibits SERCA in ASMC.

There is a need to identify novel agents that elicit small airway relaxation when b 2 -adrenoceptor agonists become ineffective in difficult-to-treat asthma. Because chronic treatment with the synthetic peroxisome proliferator activated receptor (PPAR)g agonist rosiglitazone (RGZ) inhibits airway hyperresponsiveness in mouse models of allergic airways disease, we tested the hypothesis that RGZ causes acute airway relaxation by measuring changes in small airway size in mouse lung slices. Whereas the b-adrenoceptor agonists albuterol (ALB) and isoproterenol induced partial airway relaxation, RGZ reversed submaximal and maximal contraction to methacholine (MCh) and was similarly effective after precontraction with serotonin or endothelin-1. Concentrationdependent relaxation to RGZ was not altered by the b-adrenoceptor antagonist propranolol and was enhanced by ALB. RGZ-induced relaxation was mimicked by other synthetic PPARg agonists but not by the putative endogenous agonist 15-deoxy-PGJ 2 and was not prevented by the PPARg antagonist GW9662.