Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of β2-Adrenergic Receptors
Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of β2-Adrenergic Receptors
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DOI：
10.1124/mol.114.096800
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发表时间：
2015-06-01
影响因子：
3.6
通讯作者：
Clark, Richard B.
中科院分区：
文献类型：
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作者：
Gimenez, Luis E.;Baameur, Faiza;Clark, Richard B.
沙美特罗是一种长效 β2-肾上腺素能受体 (β2AR) 激动剂，广泛用作支气管扩张剂，与类固醇联用治疗持续性哮喘和慢性阻塞性肺病。先前的研究表明，沙美特罗对腺苷酸环化酶的激活作用较弱；然而，其在 β2AR 复杂脱敏中的功效仍然知之甚少。在这项工作中，我们通过配体-β2AR与抑制蛋白结合的生物发光共振能量转移（BRET）研究以及cAMP转换的动力学研究，深入了解G蛋白偶联受体激酶/抑制蛋白和蛋白激酶A在沙美特罗介导的脱敏中所起的作用。首先，BRET 证明相对于异丙肾上腺素，沙美特罗将抑制蛋白募集到 β2AR 的功效大大降低。 5分钟刺激后BRETISO/BRETSALM的比率为20，35分钟后下降至5，反映出BRETISO逐渐下降且BRETSALM稳定。其次，为了评估沙美特罗的功能性脱敏功效，我们检查了在存在和不存在磷酸二酯酶抑制的情况下沙美特罗诱导的人气道平滑肌细胞中cAMP积累（0-30分钟）的动力学。对两种激动剂的 cAMP 周转形成的分析表明，沙美特罗具有显着的脱敏作用，尽管其相对于异丙肾上腺素有所降低。在短期（10 分钟）或长期（2 和 14 小时）沙美特罗预处理后使用异丙肾上腺素救援方案，我们发现沙美特罗逐渐抑制异丙肾上腺素刺激，但并不能阻止异丙肾上腺素随后的救援和额外的异丙肾上腺素介导的脱敏作用。我们的研究结果揭示了功能性脱敏的复杂功效，表明尽管沙美特罗对腺苷酸环化酶激活和 G 蛋白偶联受体激酶/抑制蛋白介导的脱敏作用较弱，但它在高度放大的蛋白激酶 A 介导的事件中充当强激动剂。
Salmeterol is a long-acting beta 2-adrenergic receptor (beta 2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the beta 2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-beta 2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to beta 2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.