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ß2-Adrenoceptor – cAMP signaling exerts dual effects on ß2-adrenoceptor expression in human lung fibroblasts, delayed up-regulated inhibitory factors oppose a rapid in onset, direct stimulation of gene expression Based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and COPD. However, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyperreactivity, possibly because of loss of β-adrenoceptor function. Therefore, the molecular regulation of β2-adrenoceptor expression appears to be of particular interest and was addressed here. MRC-5 human lung fibroblasts, known to express β2-adrenoceptors (Lamyel et al., 2011), were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mRNA determination by qPCR. Under control conditions the β2-adrenoceptors mRNA levels, expressed as ΔCt over GAPDH, amounted to 12.1±0.1 (mean±s.e.mean, n = 60). After inhibition of mRNA synthesis by actinomycin D (30 µM), β2-adrenoceptor mRNA decreased with a half-life of 25 min, whereas inhibition of protein synthesis by cycloheximide (30 µM) resulted in an increase of β2-adrenoceptor mRNA by 333±85%, 502±52% and 640±165% (n = 5-9, each p<0.01 vs control) within 1.5, 4 and 6.5 h, respectively. Exposure to the long-acting β2-adrenoceptor agonists formoterol or olodaterol (1-100 nM) resulted in a rapid increase in β2-adrenoceptor mRNA (maximally within 1 h by 100±19% and 110±19% at 10 nM formoterol and 10 nM olodaterol, respectively, each p<0.001 vs control). However, after 4 h exposure to 10 nM formoterol or 10 nM olodaterol a reduction in β2-adrenoceptor mRNA by 59±8% and 58±6% (each p<0.001 vs control) respectively, was observed, but prolongation of the agonist exposure time to 48 h resulted again in an increase in β2-adrenoceptor mRNA levels by 62±12% (p<0.01 vs control). Both, the time-dependent stimulatory and inhibitory effects of β2-adrenoceptor agonists were mimicked by forskolin (10 µM, increase by 88±14% (p<0.01 vs control) and inhibition by 49±4% (p<0.001 vs control)) and cholera toxin (5 ng/ml, increase by 76±12% (p<0.01 vs control) and inhibition by 77±7% (p<0.001 vs control)). The rapid formoterol-induced up-regulation of β2-adrenoceptor mRNA was blocked by the β2-adrenoceptor antagonist ICI 118551 and prevented by actinomycin D, but not by cycloheximide. Thus, after 1 h exposure to 10 nM formoterol in presence of cycloheximide, β2-adrenoceptor mRNA was increased by 750±133% (p<0.01 vs cycloheximide alone) and after 4 h exposure to 10 nM olodaterol or 10 µM forskolin in presence of cycloheximide, β2-adrenoceptor mRNA was increased by 1550±351% and 1380±275% respectively, each p<0.001 vs cycloheximide alone). 1 h exposure to the prostanoid receptor agonist butaprost (100 nM) caused also an increase in β2-adrenoceptor mRNA by 95±12% (p<0.01 vs control), and effects of olodaterol and butaprost were not additive. Likewise, the stimulatory effects of forskolin and olodaterol were also not additive. In conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level, suggesting that β2-adrenoceptor expression may rapidly respond to physiological or pathological changes as well as pharmacological interventions. The observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. Although both, the time-dependent up- and down-regulation of the β2-adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase - cAMP signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene.
Lamyel F, Warnken-Uhlich M, Seemann WK et al., (2011) Naunyn Schmiedeberg´s Arch Pharmacol. 384:133–145.
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