Expression of functional β 2-adrenergic receptors in the lung epithelial cell lines 16HBE14o −, Calu-3 and A549

Abstract

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 (−)-[ 125I]-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.