Shaikh Nooreen, Johnson Malcolm, Hall David A, Chung Kian Fan, Riley John H, Worsley Sally, Bhavsar Pankaj K
Experimental Studies, National Heart and Lung Institute, Imperial College London.
Biomedical Research Unit, Royal Brompton and Harefield NHS Trust, London.
Int J Chron Obstruct Pulmon Dis. 2017 Jun 30;12:1903-1913. doi: 10.2147/COPD.S134420. eCollection 2017.
Intracellular mechanisms of action of umeclidinium (UMEC), a long-acting muscarinic receptor antagonist, and vilanterol (VI), a long-acting β-adrenoceptor (βR) agonist, were investigated in target cells: human airway smooth-muscle cells (ASMCs).
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 ([Ca]) using a fluorescence assay, and regulator of G-protein signaling 2 (RGS2) messenger RNA using real-time quantitative polymerase chain reaction.
VI and salmeterol (10-10 M) induced cAMP production from ASMCs in a concentration-dependent manner, which was greater for VI at all concentrations. βR antagonism by propranolol or ICI 118.551 (10-10 M) resulted in concentration-dependent inhibition of VI-induced cAMP production, and ICI 118.551 was more potent. MCh (5×10 M, 30 minutes) attenuated VI-induced cAMP production (<0.05), whereas pretreatment with UMEC (10 M, 1 hour) restored the magnitude of VI-induced cAMP production. ASMC stimulation with MCh (10-5×10 M) resulted in a concentration-dependent increase in [Ca], which was attenuated with UMEC pretreatment. Reduction of MCh-induced [Ca] release was greater with UMEC + VI versus UMEC. UMEC enhanced VI-induced RGS2 messenger RNA expression.
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.
在靶细胞——人气道平滑肌细胞（ASMCs）中研究了长效毒蕈碱受体拮抗剂乌美溴铵（UMEC）和长效β-肾上腺素能受体（βR）激动剂维兰特罗（VI）的细胞内作用机制。
用VI、UMEC、UMEC与VI联合或对照化合物（沙美特罗、普萘洛尔、ICI 118.551或乙酰甲胆碱[MCh]）处理来自健康肺移植供体气管的ASMCs。使用酶联免疫吸附测定法测量环磷酸腺苷（cAMP），使用荧光测定法测量细胞内游离钙（[Ca]），并使用实时定量聚合酶链反应测量G蛋白信号调节因子2（RGS2）信使核糖核酸。
VI和沙美特罗（10⁻¹⁰ M）以浓度依赖性方式诱导ASMCs产生cAMP，在所有浓度下VI诱导的cAMP产生量均更高。普萘洛尔或ICI 118.551（10⁻¹⁰ M）对βR的拮抗作用导致VI诱导的cAMP产生受到浓度依赖性抑制，且ICI 118.551的作用更强。MCh（5×10⁻⁶ M，30分钟）减弱了VI诱导的cAMP产生（<0.05），而用UMEC（10⁻⁶ M，1小时）预处理可恢复VI诱导的cAMP产生幅度。用MCh（10⁻⁵×10⁻⁶ M）刺激ASMCs导致[Ca]浓度依赖性增加，而UMEC预处理可减弱这种增加。与单独使用UMEC相比，UMEC + VI对MCh诱导的[Ca]释放的减少作用更大。UMEC增强了VI诱导的RGS2信使核糖核酸表达。
这些数据表明，UMEC可逆转胆碱能对VI诱导的cAMP产生的抑制作用，并且与单独使用相比，UMEC与VI联合使用时是一种更有效的毒蕈碱受体拮抗剂。
