Background: Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting β2-adrenoceptor (β2R) 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. β2R 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.