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 beta(2)-adrenoceptor (beta R-2) 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. beta R-2 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 (5x10(-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)-5x10(-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.