Intracellular interactions of umeclidinium and vilanterol in human airway smooth muscle.

BACKGROUND: Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting ß2-adrenoceptor (ß2R) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs). MATERIALS AND METHODS: ASMCs from tracheas of healthy lung-transplant donors were treated with VI, UMEC, UMEC and VI combined, or control compounds (salmeterol, propranolol, ICI 118.551, or methacholine [MCh]). Cyclic adenosine monophosphate (cAMP) was measured using an enzyme-linked immunosorbent assay, intracellular free calcium ([Ca2+]i) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction. RESULTS: VI and salmeterol (10-12-10-6 M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. ß2R antagonism by propranolol or ICI 118.551 (10-12-10-4 M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10-6 M, 30 minutes) attenuated VI-induced cAMP production (P<0.05), whereas pretreatment with UMEC (10-8 M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10-11-5×10-6 M) resulted in a concentration-dependent increase in [Ca2+]i, which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca2+]i release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression. CONCLUSION: These data indicate that UMEC reverses cholinergic inhibition of VI-induced cAMP production, and is a more potent muscarinic receptor antagonist when in combination with VI versus either alone.

Impaired nuclear translocation of the glucocorticoid receptor in corticosteroid-insensitive airway smooth muscle in severe asthma.

RATIONALE: Patients with severe asthma (SA) are less responsive to the beneficial effects of corticosteroid (CS) therapy, and relative CS insensitivity has been shown in airway smooth muscle cells (ASMC) from patients with SA. OBJECTIVES: We investigated whether there was a defect in the actions of the glucocorticoid receptor (GR) underlying the ability of CS to suppress the inflammatory response in ASMC of patients with SA. ASMC from healthy subjects (n = 10) and subjects with severe (n = 8) and nonsevere asthma (N-SA; n = 8) were cultured from endobronchial biopsies. MEASUREMENTS AND MAIN RESULTS: GR expression in ASMC from SA and N-SA was reduced compared with that from healthy subjects by 49% (P < 0.01). Although baseline levels of nuclear GR were similar, GR nuclear translocation induced by dexamethasone (10(-7) M) in SA was 60% of that measured in either healthy subjects or subjects with N-SA. Tumor necrosis factor (TNF)-α induced greater nuclear factor (NF)-κB (p65) mRNA expression in ASMC from subjects with SA (5.6- vs. 2.0-fold; P < 0.01), whereas baseline and TNF-α-induced nuclear translocation and dexamethasone-mediated suppression of p65 expression were similar between groups. Dexamethasone, although not modulating TNF-α-induced p65 nuclear translocation, attenuated p65 recruitment to the CCL11 promoter in the healthy and N-SA groups, but this suppressive effect was impaired in subjects with SA. CONCLUSIONS: Decreased GR expression with impaired nuclear translocation in ASMC, associated with reduced dexamethasone-mediated attenuation of p65 recruitment to NF-κB-dependent gene promoters, may underlie CS insensitivity of severe asthma.

The Meis homeoprotein regulates the axolotl Prod 1 promoter during limb regeneration.

During limb regeneration in salamanders the blastemal cells give rise only to structures distal to the level of amputation. This proximodistal identity can be regulated by ectopic expression of Meis homeoproteins or the three finger protein Prod 1 which acts at the cell surface. It has been suggested that Meis acts by regulating the transcription of Prod 1. We have sequenced the axolotl Prod 1 promoter and selected two candidate sites for binding Meis homeoproteins. The sites were mutated in various combinations in promoters expressing a luciferase reporter gene. The promoter activity was assayed by nucleofecting AL1 cells, a cultured axolotl limb cell line that expresses both Prod 1 and Meis 1 and 2. The activity of the promoter was inhibited by 60% after mutation at Meis site 1, but not at Meis site 2. The promoter constructs were electroporated into axolotl limb blastemas and the wild type promoter was more active in a proximal blastema than in a contralateral distal blastema. The wild type promoter was significantly more active than a (site 1+site 2) mutant promoter in contralateral proximal blastemas, but the promoters were equivalent in contralateral distal blastemas. The separate site 1 or site 2 mutants were not significantly different from wild type in contralateral proximal blastemas, thus contrasting with the site 1 results in AL1 cells. These data provide strong support for the hypotheses that the Prod 1 promoter is regulated on the proximodistal axis, and that Meis homeoproteins directly regulate the promoter on this axis during limb regeneration in addition to cultured cells.