Identification and characterization of β-adrenergic receptors in isolated primary equine tracheal epithelial cells

Abstract

Responses and functions of airway epithelial cells are stimulated by β 2-agonists via the β 2-adrenergic receptors (β 2-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 β 1-selective (CGP 20712A) and β 2-selective (ICI 118.551) adrenergic receptor antagonists demonstrated that the β 2-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.