Mareille Warnken

Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts

In lung fibroblasts, proliferation is inhibited by activation of EP2 prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP2 receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [3H]-thymidine incorporation. The selective EP2 receptor agonist butaprost inhibited [3H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2′-O-Me-cAMP and Sp-8-pCPT-2′-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [3H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [3H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP2 receptor activation.

Pulmonary fibroblasts, an emerging target for anti-obstructive drugs

Fibrotic alterations are part of the airway re-modelling processes observed in asthma and chronic obstructive pulmonary disease. There is increasing evidence that in addition to acute bronchodilatory effects, classical anti-obstructive drugs such as muscarinic antagonists and β-adrenoceptor agonists may also modulate long-term re-modelling processes. The present review aims to summarise muscarinic and β-adrenergic effects on pulmonary fibroblasts. Recent experimental evidence demonstrated muscarinic stimulatory effects on pulmonary fibroblasts, and long-term blockade of these pro-fibrotic effects may contribute to the beneficial effects of muscarinic antagonists, as observed particularly for the long-acting muscarinic antagonist tiotropium. On the other hand, β-adrenoceptor agonists, via activation of adenylyl cyclase, can also exert various inhibitory effects on pulmonary fibroblasts, and these anti-fibrotic effects are mimicked by other agents that cause an increase in intracellular cyclic adenosine monophosphate (cAMP), such as phosphodiesterase inhibitors or EP2 prostanoid receptor agonists. In addition, the role of the extracellular signal-regulated kinase–mitogen-activated protein kinase pathway, protein kinase A and exchange protein activated by cAMP (Epac) and potential interactions between these cellular signalling pathways are discussed.

Pro-fibrotic processes in human lung fibroblasts are driven by an autocrine/paracrine endothelinergic system.

Since endothelin (ET) may act as pro‐fibrotic mediator, expression and release of ET isoforms, their receptors and potential pro‐fibrotic ET effects were studied in human lung fibroblasts.

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.

Endothelin-1 enhances β2-adrenoceptor gene transcription in human lung fibroblasts

AIMS The present study aimed to explore possible effects of endothelin-1 (ET-1) on ß(2)-adrenoceptor gene transcription in human lung fibroblasts. MAIN METHODS MRC-5 human lung fibroblasts were cultured in absence or presence of test substances, followed by ß(2)-adrenoceptor mRNA determination by quantitative real time PCR. KEY FINDINGS ET-1 caused a marked and rapid in onset (1 hr) increase in β(2)-adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the β(2)-adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on β(2)-adrenoceptor mRNA was prevented by the non-selective ET-A/ET-B receptor antagonist bosentan, indicating that it was mediated via specific ET receptors. In the presence of actinomycin D the effect of ET-1 was prevented indicating that ET-1 acts via increased transcription of the β(2)-adrenoceptor gene. ET-1-induced up-regulation of β(2)-adrenoceptor mRNA was also seen in the presence of cycloheximide excluding indirect effects via up-regulation of other regulatory proteins. CONCLUSIONS ET-1 can up-regulate β-adrenoceptor gene transcription in human lung fibroblasts.