BACKGROUND AND PURPOSEArtificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such 'superagonism' has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a 'superagonist'. EXPERIMENTAL APPROACHSignalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. KEY RESULTSIn CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y104 3.33 A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. 'Superagonism' is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure-signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that 'superagonism' of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. CONCLUSION AND IMPLICATIONSSupraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR 'superagonism' is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators.

Fibrosis is part of airway remodelling observed in bronchial asthma and COPD. Pro-fibrotic activity of lung fibroblasts may be suppressed by β-adrenoceptor activation. We aimed, first, to characterise the expression pattern of β-adrenoceptor subtypes in human lung fibroblasts and, second, to probe β-adrenoceptor signalling with an emphasis on anti-fibrotic actions. Using reverse transcription PCR, messenger RNA (mRNA) encoding β 2 -adrenoceptors was detected in MRC-5, HEL-299 and primary human lung fibroblasts, whereas transcripts for β 1 -and β 3 -adrenoceptors were not found. Real-time measurement of dynamic mass redistribution in MRC-5 cells revealed β-agonistinduced G s -signalling. Proliferation of MRC-5 cells (determined by [ 3 H]-thymidine incorporation) was significantly inhibited by β-agonists including the β 2 -selective agonist formoterol (−logIC 50 , 10.2) and olodaterol (−logIC 50 , 10.6). Formoterol's effect was insensitive to β 1 -antagonism (GCP 20712, 3 μM), but sensitive to β 2 -antagonism (ICI 118,551; apparent, pA 2 , 9.6). Collagen synthesis in MRC-5 cells (determined by [ 3 H]-proline incorporation) was inhibited by β-agonists including formoterol (−logIC 50 , 10.0) and olodaterol (−logIC 50 , 10.3) in a β 2 -blockersensitive manner. α-Smooth muscle actin, a marker of myofibroblast 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 β 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 β 2 -agonist treatment of bronchial asthma and COPD.

BACKGROUND AND PURPOSESince 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. EXPERIMENTAL APPROACHMRC-5 and primary human lung fibroblasts (phLFb) were cultured. Expression of prepro-ET isoforms was determined by qPCR and release of ET-1 by ELISA. ET receptor function was analysed by real-time measurement of dynamic mass redistribution (DMR). Incorporation of [ KEY RESULTSET-1 is the predominant ET in human lung fibroblasts (hLF), and TGF-b caused a further, selective and sustained up-regulation of ET-1 resulting in increased extracellular ET-1 accumulation. hLFb express mRNA encoding ET-A and ET-B receptors. Expression of both receptors was confirmed at protein level. ET-1 induced marked DMR signals, an effect that involved ET-A and ET-B receptors. Stimulatory effects of ET-1 on hLFb proliferation and collagen synthesis were mediated exclusively via ET-A receptors. ET-1, again via ET-A receptors, induced rapid activation of ERK MAPK, shown to be a crucial cellular signal in ET-1-induced collagen synthesis. ET-1-induced activation of ERK and collagen synthesis was, in contrast to corresponding effect of a muscarinic agonist, largely insensitive to pertussis toxin. CONCLUSIONS AND IMPLICATIONShLFb are endowed with all elements necessary to build a functional autocrine/paracrine endothelinergic system, which appears to drive pro-fibrotic airway and lung remodelling processes, effects for which only ET-A, but not ET-B receptors appear to be of significance.