University of Leipzig, Faculty of Veterinary Medicine, Institute of Pharmacology, Pharmacy and Toxicology, An den Tierkliniken 15, 04103 Leipzig, Germany.
Pulm Pharmacol Ther. 2011 Feb;24(1):174-81. doi: 10.1016/j.pupt.2010.06.003. Epub 2010 Jun 25.
Responses and functions of airway epithelial cells are stimulated by β₂-agonists via the β₂-adrenergic receptors (β₂-ARs)-G(s)-protein-cAMP-system, thus, affecting airway inflammation such as in asthma and equine recurrent airway obstruction (RAO). Though horses can be used as large animal model for human asthma, evaluation of the expression and functions of the β-AR system in primary equine airway epithelial cells has not been yet carried out. Thus, for the first time, we determined the β-AR density and subtype distribution by [¹²⁵I]-iodocyanopindolol (ICYP) binding, examined β-AR function by cAMP assay as well as their expression by western blot analysis and immunocytochemical staining in primary equine tracheal epithelial cells (ETEC). Cells were collected from 19 horses and cultured subsequently. The specific ICYP binding was saturable and of high affinity: in freshly isolated cells the receptor density (B(max)) and ICYP affinity (K(D)) for β-ARs were 12727 ± 883 binding sites/cell and 31.78 ± 6.57 pM, respectively, and in cultured ETEC 3730 ± 212 binding sites/cell and 15.26 ± 3.37 pM, respectively. The β-AR subtype assessed by β₁-selective (CGP 20712A) and β₂-selective (ICI 118.551) adrenergic receptor antagonists demonstrated that the β₂-AR subtype predominated (>95%) in both cell populations (p < 0.001). The β-AR agonists increased cAMP formation with a rank order of potency: isoproterenol > epinephrine > norepinephrine. ICI 118.551 (100 nM) significantly blocked (p < 0.05) isoproterenol-induced cAMP accumulation but not CGP 20712A (300 nM). Western blot analyses and immunocytochemical staining further indicated the expression of the β(2)-AR subtype in both cell preparations. Our data indicate that in acutely dissociated and primary cultured ETEC the β(2)-AR-AC system is expressed, but varies considerably between the two preparations.
气道上皮细胞的反应和功能受到β₂-激动剂通过β₂-肾上腺素能受体（β₂-ARs）-G(s)-蛋白-cAMP 系统的刺激，从而影响气道炎症，如哮喘和马复发性呼吸道阻塞（RAO）。虽然马可以作为人类哮喘的大型动物模型，但尚未对原代马气道上皮细胞中β-AR 系统的表达和功能进行评估。因此，我们首次通过[¹²⁵I]-碘氰基苯氧丙醇（ICYP）结合来确定β-AR 密度和亚型分布，通过 cAMP 测定法检查β-AR 功能，以及通过 Western blot 分析和免疫细胞化学染色来检查其在原代马气管上皮细胞（ETEC）中的表达。细胞取自 19 匹马并随后进行培养。特异性 ICYP 结合是饱和的且具有高亲和力：在新鲜分离的细胞中，β-AR 的受体密度（B（max））和 ICYP 亲和力（K（D））分别为 12727±883 个结合位点/细胞和 31.78±6.57 pM，而在培养的 ETEC 中为 3730±212 个结合位点/细胞和 15.26±3.37 pM。通过β₁-选择性（CGP 20712A）和β₂-选择性（ICI 118.551）肾上腺素能受体拮抗剂评估的β-AR 亚型表明，β₂-AR 亚型在两种细胞群体中均占优势（p <0.001）。β-AR 激动剂的效力递增顺序增加 cAMP 的形成：异丙肾上腺素>肾上腺素>去甲肾上腺素。ICI 118.551（100 nM）显著阻断（p <0.05）异丙肾上腺素诱导的 cAMP 积累，但 CGP 20712A（300 nM）不阻断。Western blot 分析和免疫细胞化学染色进一步表明，两种细胞制剂均表达β（2）-AR 亚型。我们的数据表明，在急性分离和原代培养的 ETEC 中表达了β（2）-AR-AC 系统，但两种制剂之间存在很大差异。
