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.

ß2-adrenoceptors and muscarinic receptors mediate opposing effects on endothelin-1 expression in human lung fibroblasts.

Human lung fibroblasts are a potential source of endothelin-1 (ET-1), a pro-fibrotic mediator. The present study explored possible muscarinic and ß-adrenergic modulations of ET-1 expression in human lung fibroblasts. MRC-5 human lung fibroblasts were cultured. Expression of prepro-endothelin-1 (ppET-1) mRNA was determined by quantitative real time PCR. [(3)H]-Proline incorporation was determined as measure of collagen synthesis. The muscarinic agonist oxotremorine induced, in a tiotropium-sensitive manner, a three-fold increase in ppET-1 mRNA. The ß(2)-adrenoceptor agonist olodaterol caused a reduction of ppET-1 mRNA by 45%. Olodaterol also opposed the stimulatory effect of oxotremorine. The effect of olodaterol was mimicked by the protein kinase A agonist 6-Bnz-cAMP, whereas the Epac (exchange protein activated by cAMP) agonist 8-CPT-2'-O-Me-cAMP was less effective. Transforming growth factor-ß(1) (TGF-ß, 0.3 and 1 ng/ml) induced a three- and eight-fold increase in pp-ET-1 mRNA, respectively. Olodaterol opposed the effect of 0.3, but not that of 1 ng/ml TGF-ß. Likewise, 6-Bnz-cAMP opposed the effect of 0.3, but not that of 1 ng/ml TGF-ß. TGF-ß inhibited ß(2)-adrenoceptor mRNA expression, maximally by 90%. Muscarinic agonist-induced stimulation of [(3)H]-proline incorporation was attenuated by the endothelin ET1 receptor antagonist bosentan. In conclusion, ET-1 expression in human lung fibroblasts is regulated by stimulatory muscarinic receptors and inhibitory ß(2)-adrenoceptors. Since muscarinic up-regulation of ET-1 contributes to pro-fibrotic effects of muscarinic stimuli, inhibition of ET-1 expression could contribute to long-term beneficial effects of long-acting ß(2)-adrenoceptor agonists and long-acting muscarinic antagonists. However, excessive exposure to TGF-ß results in loss of ß-adrenoceptor expression and function of its down-stream signaling.

Endothelin-1 enhances ß2-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 ß(2)-adrenoceptor mRNA, an effect additive to that of short time (1 hr) exposure to the ß(2)-adrenoceptor agonist olodaterol. The stimulatory effect of ET-1 on ß(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 ß(2)-adrenoceptor gene. ET-1-induced up-regulation of ß(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 ß-adrenoceptor gene transcription in human lung fibroblasts.

Insulin action on H292 bronchial carcinoma cells as compared to normal bronchial epithelial cells.

Inhaled insulin may contribute to bronchial carcinoma due to IGF-I receptor activation by high local concentrations. Therefore, effects of insulin and IGF-I on human bronchial carcinoma cells (H292) and normal bronchial epithelium cells (HBE) were studied. TGF-ß was included since it also influences carcinoma progression. H292 and HBE cells expressed both the insulin receptor and the IGF-I receptor; in H292 cells an additional, shorter, splicing variant (IR-A) of the insulin receptor was present. Insulin receptor expression was around four to five times higher in H292 than in HBE cells at mRNA and protein levels. Insulin and TGF-ß exerted contrary actions on proliferation and gene expression in H292 cells. Genes regulated by insulin, IGF-I, and TGF-ß were linked to inflammation, cell adhesion, muscle contraction and differentiation. Insulin and IGF-I also suppressed DNA repair genes. EC(50) for insulin-induced proliferation was around 5 nM in H292 and around 30 nM HBE cells. The EC(50) values for gene expression ranged from 9 to 90 nM in both cell types, dependent on the gene studied. In H292 cells, the proliferative response was much stronger if TGF-ß was present. In HBE cells this interaction of insulin and TGF-ß was not observed, and changes in gene expression were mostly lower by at least 10-fold as compared to H292. All in all, the insulin effects in H292 were generally much stronger than in HBE cells and - with regard to proliferation - occurred at lower concentrations. Thus, insulin will hardly induce cancer from normal bronchial cells but may favour progression of pre-existing tumours.

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 beta-adrenoceptor agonists may also modulate long-term re-modelling processes. The present review aims to summarise muscarinic and beta-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, beta-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.

Inhibition of NADPH oxidase by apocynin inhibits lipopolysaccharide (LPS) induced up-regulation of arginase in rat alveolar macrophages.

Reactive oxygen species participate in the pathogenesis of inflammatory airway diseases, in which increased arginase may play a role by interfering with nitric oxide (NO) synthesis and providing substrate for collagen synthesis. Therefore a modulatory role of reactive oxygen species for arginase was explored in alveolar macrophages using the NADPH oxidase inhibitor apocynin. The effects of lipopolysacharides (LPS) and apocynin on nitrite accumulation, arginase activity and mRNA for inducible NO synthase (iNOS), arginase I and II were determined. Superoxide anion (O(2)(-)) release was analysed by the iodonitrotetrazolium (INT) formazan assay. LPS (1 microg/ml) caused a 55%, transient increase in INT formation, i.e. O(2)(-) release which was inhibited by apocynin (500 microM). LPS caused a 2 fold increase in arginase activity and a marked increase in mRNA encoding arginase I, the predominant isoenzyme. Both effects were largely attenuated by apocynin. Apocynin did not affect the stability of arginase I mRNA, but accelerated the decline of arginase activity when protein synthesis was inhibited by cycloheximide. Apocynin also reduced LPS-induced nitrite accumulation (by 30%) and iNOS mRNA expression, but the magnitude of these effects was smaller than that on arginase I. Arginase I mRNA was also increased following exposure to hydrogen peroxide (H(2)O(2), 200 muM). In conclusion, inhibition of NADPH oxidase in alveolar macrophages causes down-regulation of arginase, indicating that reactive oxygen species exert stimulatory effects on arginase. Enhanced transcription of arginase mRNA and prolongation of the life time of the active enzyme appear to contribute to the enhanced arginase activity.