Distinct PKA and Epac compartmentalization in airway function and plasticity

<p>Asthma and chronic obstructive pulmonary disease (COPD) are obstructive lung diseases characterized by airway obstruction, airway inflammation and airway remodelling. Next to inflammatory cells and airway epithelial cells, airway mesenchymal cells, including airway smooth muscle cells and (myo)fibroblasts, substantially contribute to disease features by the release of inflammatory mediators, smooth musde contraction, extracellular matrix deposition and structural changes in the airways. Current pharmacological treatment of both diseases intends to target the dynamic features of the endogenous intracellular suppressor cyclic AMP (cAMP). This review will summarize our current knowledge on cAMP and will emphasize on key discoveries and paradigm shifts reflecting the complex spatio-temporal nature of compartmentalized cAMP signalling networks in health and disease. As airway fibroblasts and airway smooth muscle cells are recognized as central players in the development and progression of asthma and COPD, we will focus on the role of cAMP signalling in their function in relation to airway function and plasticity. We will recapture on the recent identification of cAMP-sensing multi-protein complexes maintained by cAMP effectors, including A-kinase anchoring proteins (AKAPs), proteins kinase A (PICA), exchange protein directly activated by cAMP (Epac), CAMP-elevating seven-transmembrane (7TM) receptors and phospho-diesterases (PDEs) and we will report on findings indicating that the pertubation of compartmentalized cAMP signalling correlates with the pathopysiology of obstructive lung diseases. Future challenges include studies on CAMP dynamics and compartmentalization in the lung and the development of novel drugs targeting these systems for therapeutic interventions in chronic obstructive inflammatory diseases. (C) 2012 Elsevier Inc. All rights reserved.</p>

Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells

Oenema TA, Smit M, Smedinga L, Racke K, Halayko AJ, Meurs H, Gosens R. Muscarinic receptor stimulation augments TGF-beta(1)-induced contractile protein expression by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L589-L597, 2012. First published August 3, 2012; doi:10.1152/ajplung.00400.2011.-Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-beta(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M-2 and M-3 receptors were stimulated with methacholine (MCh), TGF-beta(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-alpha-actin and calponin by TGF-beta(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-alpha-actin stress fibers by TGF-beta(1). MCh did not augment TGF-beta(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-beta(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3 beta and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-beta(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD

The beta(2)-subtype of adrenoceptors mediates inhibition of pro-fibrotic events in human lung fibroblasts

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by beta-adrenoceptor activation. We aimed, first, to characterise the expression pattern of beta-adrenoceptor subtypes in human lung fibroblasts and, second, to probe beta-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding beta(2)-adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for beta(1)- and beta(3)-adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed beta-agonist-induced G(s)-signalling. Proliferation of MRC-5 cells (determined by [H-3]-thymidine incorporation) was significantly inhibited by beta-agonists including the beta(2)-selective agonist formoterol (-logIC(50), 10.2) and olodaterol (-logIC(50), 10.6). Formoterol's effect was insensitive to beta(1)-antagonism (GCP 20712, 3 mu M), but sensitive to beta(2)-antagonism (ICI 118,551; apparent, pA (2), 9.6). Collagen synthesis in MRC-5 cells (determined by [H-3]-proline incorporation) was inhibited by beta-agonists including formoterol (-logIC(50), 10.0) and olodaterol (-logIC(50), 10.3) in a beta(2)-blocker-sensitive manner. alpha-Smooth muscle actin, a marker of myo-fibroblast differentiation, was down-regulated at the mRNA and the protein level by about 50% following 24 and 48 h exposure to 1 nM formoterol, a maximally active concentration. In conclusion, human lung fibroblasts exclusively express beta(2)-adrenoceptors and these mediate inhibition of various markers of pro-fibrotic cellular activity. Under clinical conditions, anti-fibrotic actions may accompany the therapeutic effect of long-term beta(2)-agonist treatment of bronchial asthma and COPD

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor

Prostanoid receptors of the EP(3) subtype mediate inhibition of evoked [(3)H]acetylcholine release from isolated human bronchi

1. The release of neuronal [(3)H]acetylcholine (ACh) from isolated human bronchi after labelling with [(3)H]choline was measured to investigate the effects of prostanoids. 2. A first period of electrical field stimulation (S(1)) caused a [(3)H]ACh release of 320±70 and 200±40 Becquerel (Bq) g(−1) in epithelium-denuded and epithelium-containing bronchi respectively (P>0.05). Subsequent periods of electrical stimulation (S(n), n=2, 3, and 4) released less [(3)H]ACh, i.e. decreasing S(n)/S(1) values were obtained (0.76±0.09, 0.68±0.07 and 0.40±0.04, respectively). 3. Cumulative concentrations (1–1000 nM) of EP-receptor agonists like prostaglandin E(2), nocloprost, and sulprostone (EP(1) and EP(3) selective) inhibited evoked [(3)H]ACh release in a concentration dependent manner with IC(50) values between 4–14 nM and maximal inhibition of about 70%. 4. The inhibition of evoked [(3)H]ACh release by prostaglandin E(2), nocloprost and sulprostone was not affected by the DP-, EP(1)- and EP(2)-receptor antagonist AH6809 at a concentration of 3 μM, i.e. a 3–30 times greater concentration than its affinity (pA(2) values) at the respective receptors. 5. Circaprost (IP-receptor agonist; 1–100 nM), iloprost (IP- and EP(1)-receptor agonist; 10-1000 nM) and U-46619 (TP-receptor agonist; 100–1000 nM) did not significantly affect [(3)H]ACh release. 6. Blockade of cyclooxygenase by 3 μM indomethacin did not significantly modulate evoked [(3)H]ACh release in epithelium-containing and epithelium-denuded bronchi. Likewise, the combined cyclo- and lipoxygenase inhibitor BW-755C (20 μM) did not affect evoked [(3)H]ACh release. 7. In conclusion, applied prostanoids appear to inhibit [(3)H]ACh release in epithelium-denuded human bronchi under the present in vitro conditions, most likely via prejunctional prostanoid receptors of the EP(3) subtype