Characterization of human and rodent native and recombinant adenosine A(2B) receptors by radioligand binding studies

Abstract

Adenosine A(2B) receptors of native human and rodent cell lines were investigated using [(3)H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [(3)H]PSB-298 showed saturable and reversible binding. It exhibited a K(D) value of 60 +/- 1 nM and limited capacity (B(max) = 3.511 fmol per milligram protein) at recombinant human adenosine A(2B) receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5'-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A(2B) receptors. Adenosine A(2B) receptors were shown to be the major adenosine A(2) receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [(3)H]PSB-298 is a selective radioligand for adenosine A(2B) binding sites in this cell line.

Figure 1

Structures of antagonist radioligands used for adenosine A_2B receptor binding assays

Figure 2

Preparation of [³H]PSB-298 from the propargyl precursor PSB-297 by catalytic hydrogenation (* denotes position of radiolabel)

Figure 3

Electropherogram of [³H]PSB-298 in the presence of PSB-297 as an internal standard. The separation conditions were 150 mM Tris-HCl, 100 mM sodium dodecyl sulfate, pH 9.1, fused silica capillary, 37 cm length (30 cm to the detector), 75 µM I.D.; 10 kV; 25°C; detection at 321 nm; pressure injection (0.5 p.s.i., 5 s)

Figure 4

UV spectrum of PSB-298 (at a concentration of 0.01 mg/ml)

Figure 5

Kinetics of [³H]PSB-298 binding (1 nM) to membranes recombinantly expressing the human adenosine A_2B receptor at 25°C. A Association curve, B dissociation curve. Dissociation was initiated by the addition of 1 mM NECA (final concentration) after 180 min of pre-incubation. Experiments revealed a kinetic K_D value of 25 nM. Data points represent means from a typical experiment performed in duplicates

Figure 6

Saturation experiment of [³H]PSB-298 binding to human adenosine A_2B receptors recombinantly expressed in HEK-293 cells. The Scatchard-Rosenthal plot of the saturation binding experiment visualizes the presence of one single class of binding sites. Total binding (▪), specific binding (•), nonspecific binding (≆)

Figure 7

Effects of different concentrations of sodium chloride on radioligand binding to recombinant human adenosine A_2B receptors expressed in HEK-293 cell membranes. Membranes (50 µg) were incubated for 60 min at 25°C with 1 nM [³H]PSB-298. Non-specific binding was determined with 1 mM NECA. The data are means of four independent experiments ± SEM

Figure 8

Effect of sodium chloride (100 mM) on the binding of the antagonist PSB-298 and the agonist NECA, respectively, versus the radioligand [³H]PSB-298. Sodium shift experiments were performed at human A_2B adenosine receptors expressed in HEK-293 cell membranes with 1 nM [³H]PSB-298 and 50 µg of protein

Figure 9

Competition experiments of the antagonists caffeine, theophylline, alloxazine and CGS-15943 (a) and the agonists CADO, CPA and NECA (b) versus 1 nM [³H]PSB-298 performed at human recombinant adenosine A_2B receptors expressed in HEK-293 cells

Figure 10

Saturation of [³H]PSB-298 binding to native adenosine A_2B receptors in NG108-15 cells. The curve is representative of a single experiment from four independent experiments performed in duplicate

Figure 11

Inhibition of [³H]PSB-298 binding to INS-1 cells by CHA, CGS-21680 and Cl-IB-MECA. INS-1 cells were incubated for 80 min at 22°C in 200 µl KRH buffer containing 5.6 mM glucose, 20 nM [³H] PSB-298, and increasing concentrations of indicated compounds. Results are expressed as percent of maximum specifically bound radioactivity (non-specific binding in the presence of 1 mM unlabeled NECA was subtracted). Each value represents the mean ± SEM of five separate experiments

Figure 12

Inhibition of [³H]PSB-298 binding to INS-1 cells by PSB- 53 and PSB-1115. INS-1 cells were incubated for 80 min at 22°C in 200 µl KRH buffer containing 5.6 mM glucose, 20 nM [³H] PSB-298, 1 mM CHA (adenosine A₁ receptor agonist) and increasing concentrations of indicated compounds (adenosine A_2B receptor antagonists). Results are expressed as percent of maximum specifically bound radioactivity (non-specific binding in the presence of 1 mM unlabeled NECA was subtracted). Each value represents the mean ± SEM of five separate experiments