Gene dose effect reveals no Gs-coupled A2A adenosine receptor reserve in murine T-lymphocytes: studies of cells from A2A-receptor-gene-deficient mice.

Agonist binding to extracellular A2A adenosine receptors (A2ARs) inhibits the activation of virtually all tested functions of T-cells and can induce apoptosis in thymocytes. The evaluation of levels of expression of these immunosuppressive receptors is expected to clarify whether the absence of spare A2ARs (no 'receptor reserve') might be one of the mechanisms of attenuation of the effects of extracellular adenosine on T-cells. A2A transcript is found in T-cells and functional receptors can be demonstrated, but the density of receptor on T-cells is too low to be detected by radioligand binding. Studies of direct radioligand binding to murine brain with the selective A2AR agonist [3H]CGS21680 (2-(4-[(2-carboxyethyl)-phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosine) established that striata levels of A2AR are virtually absent from A2A knock-out mice. Mice that are heterozygous (A2AR+/-) for the A2AR express significantly decreased levels of A2AR. To test for the presence of spare receptors in T-cells we took advantage of this gene dose effect and examined whether the decrease in the number of receptors in thymocytes from A2AR+/- mice was proportionately reflected in a decrease in the functional cAMP response of T-cells to adenosine. cAMP accumulation and apoptosis induced by adenosine and by A2AR agonist are of a lower magnitude in T-cells from A2AR+/- heterozygous mice than in T-cells from A2AR+/+ littermate control mice. These results indicate that there is no A2AR reserve in murine T-cells. Strongly decreased adenosine-triggered cAMP increases were detected in thymocytes from A2AR-/- mice, suggesting that A2B adenosine receptors cannot fully compensate for the loss of A2ARs in murine T-cells. We conclude that the number of A2ARs is the limiting factor in determining the maximal cAMP response of T-lymphocytes to extracellular adenosine, thereby minimizing the immunosuppressive effects of extracellular adenosine.

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.

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).