Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549.
Adrenergic drugs acting through the beta(2)-adrenoceptor (beta(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 beta(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, [(3)H]-radioimmunoassay and immunocytochemical staining. After 4-5 days in culture, all three cell types produced beta(2)-AR mRNA and protein at a magnitude of gene expression levels Calu-3>or=16HBE14o(-)>A549, whereas control cells Cos-1 and Caco-2 were negative. The beta(2)-AR adenylate cyclase system was highly expressed and functional in the human airway epithelial cells Calu-3 and 16HBE14o(-). The mean beta(2)-AR density (B(max)), equilibrium dissociation constant (K(D)), and the percentage of beta-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 beta(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 beta(2)-selective antagonist ICI 118,551, but not by the beta(1)-selective antagonist CGP 20712A, confirming functional coupling of the beta(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 beta(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 beta-adrenoceptor signalling in the respiratory system.
Abraham
Getu
G
Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 15, D-04103 Leipzig, Germany. gabraham@rz.uni-leipzig.de
Kneuer
Carsten
C
Ehrhardt
Carsten
C
Honscha
Walther
W
Ungemach
Fritz R
FR
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000378:metabolism; D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020120:Iodocyanopindolol / Q000378:metabolism; D007545:Isoproterenol / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011869:Radioligand Assay; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D020033:Protein Isoforms; D018343:Receptors, Adrenergic, beta-2; D020120:Iodocyanopindolol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D007545:Isoproterenol