title: Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.
abstract: Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) >> N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).
authors.affiliation: Department of Internal Medicine, University of Virginia, Charlottesville, Virginia, USA. jlinden@virginia.edu
authors.forename: J
authors.initials: J
authors.lastname: Linden
authors.forename: T
authors.initials: T
authors.lastname: Thai
authors.forename: H
authors.initials: H
authors.lastname: Figler
authors.forename: X
authors.initials: X
authors.lastname: Jin
authors.forename: A S
authors.initials: AS
authors.lastname: Robeva
mesh_terms: D000818:Animals; D018927:Anti-Asthmatic Agents / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism; D058906:Purinergic P1 Receptor Agonists; D011869:Radioligand Assay; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D013806:Theophylline / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D018927:Anti-Asthmatic Agents; D058906:Purinergic P1 Receptor Agonists; D043704:Receptor, Adenosine A2B; D018047:Receptors, Purinergic P1; D014970:Xanthines; C086749:BW A522; D013806:Theophylline; C034347:enprofylline; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium