Expression of functional β_2-adrenergic receptors in the lung epithelial cell lines 16HBE14o~-, Calu-3 and A549

摘要

Adrenergic drugs acting through the β_2-adrenoceptor (β_2-AR) adenylate cyclase (AC) signal transduction system elicit a variety of responses within the mammalian airway epithelium; however, its composition of multiple phenotypically differentiated cell types complicates the understanding of the regulation cascades within this tissue. The present study evaluates β_2-AR mRNA level, number, subtype and the cyclic adenosine-3',5'-monophosphate (cyclic AMP) response to isoproterenol (iso) in the human airway epithelial cell lines 16HBE14o~-, Calu-3 and A549, using reverse transcriptase polymerase chain reaction (RT-PCR), radioligand binding studies, [~3H]-radioimmunoassay and immunocytochemical staining. After 4-5 days in culture, all three cell types produced β_2-AR mRNA and protein at a magnitude of gene expression levels Calu-3 ≥ 16HBE14o~- > A549, whereas control cells Cos-1 and Caco-2 were negative. The β_2-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o~-. The mean β_2-AR density (B_(max)), equilibrium dissociation constant (K_D), and the percentage of β-AR subtypes assessed by radioligand binding were approximately 9908 ± 1127 and 6423 ± 895 binding sites/cell, 32 ± 2.7 pM and 25 ± 1.1 pM, and approximately 100% in Calu-3 and 16HBE14o~- cells, respectively. However, in the alveolar cell type A549 the cell surface β_2-AR was virtually undetectable by (-)-[~(125)I]-iodocyanopindolol (ICYP) binding. Stimulation of cultured cells with (-)-isoproterenol enhanced the basal cyclic AMP accumulation only in Calu-3 and 16HBE14o~- cells, which was blocked by the β_2-selective antagonist ICI 118,551, but not by the β_1-selective antagonist CGP 20712A, confirming functional coupling of the β_2-AR to adenylate cyclase in these cells. Immunocytochemical staining localised the receptor on the cell membrane and the cytoplasm in Calu-3 and 16HBE14o~- cells, while it was confined to the cytoplasm only in A549 cells. In conclusion, the β_2-AR expression and its functional coupling to adenylyl cyclase was very high in the human airway epithelial cells Calu-3 and 16HBE14o~-, but not in A549, suggesting that the cell lines Calu-3 and 16HBE14o~- present suitable models to study function and regulation of the β-adrenoceptor signalling in the respiratory system.