Chemical modification and irreversible inhibition of striatal A2a adenosine receptors

Abstract

The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mM, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mM hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at greater than 5 mM inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was greater than 50 mM. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nM) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.

Fig. 1

Dose-dependent inhibition by DEP of radioligand binding at A₂- adenosine receptors in rabbit striatal membranes. Membranes were incubated (three experiments) with 5 nm [³H]CGS 21680 (●) or 1 nm [³H] XAC in the presence of 50 nm CPX (0). The preincubation was carried out for 15 min at 25°, and the subsequent binding assay involved a 60-min incubated by rapid filtration. The effect on [³H]XAC binding of 150 mm sodium chloride (×) present during the DEP preincubation is also shown.

Fig. 2

Representative saturation curve (A) and Scatchard plot (B) for the binding of [³H]CGS 21680 to A₂-adenosine receptors in rabbit striatal membranes, in the absence (○) or presence (●) of DEP (2.5 mm) in a 20-min preincubation at 25°. Membranes were washed three times with buffer (Tris, pH 7.4) at 4° before radioligand binding. Membranes were incubated with radioligand at 25° for 90 min.

Fig. 3

Representative saturation curve (A) and Scatchard plot (B) for the binding of [³H]XAC, in the presence of 50 nm CPX, to A₂-adenosine receptors in rabbit striatal membranes, in the absence (○) or presence (●) of DEP (5.0 mm) in a 20-min preincubation at 25°. Membranes were washed three times with buffer (Tris, pH 7.4) at 4° before radioligand binding. Membranes were incubated with radioligand at 25° for 1 hr.

Fig. 4

Representative inhibition curves for the displacement by NECA of [³H]XAC in the presence of 50 nm CPX binding to rabbit striatal A₂-adenosine receptors. Binding was carried out after a 20-min preincubation at 25° in the presence of 5.0 mm DEP (○) and in control membranes (●). Membranes were washed three times with buffer (Tris, pH 7.4) at 4° before radioligand binding. Membranes were incubated with radioligand at 25° for 1 hr.

Fig. 5

Effects of exposure of rabbit striatal membranes to chemical agents. Varying concentrations of sodium dithionite(●), dithiothreitol ○, mercaptoethanol (×), and hydroxylamine ■ were present during a 20-min preincubation at 25°, followed immediately by [³H]XAC(l nm)binding at A₂ -adenosine receptors (in the presence of 50 nm CPX).

Fig. 6

Radioligand binding at A₁ ([³H]PIA) and A₂ ([³H]XAC and [³H]CGS 21680) adenosine receptors in rabbit striatal membranes after a 20-min treatment at 25° with 2.5 mm levels of a non-site-directed inhibitor, DEP (■), or 100 nm levels of an A₂-selective site-directed affinity label, m-DITC-APEC (1) (■) [³H]XAC and [³H]PIA were present at a concentration of 1 nm and [³H]CGS 21680 was present at a concentration of 5 nm(three experiments).

Fig. 7

Saturation curves for the binding of [³H]CGS 21680 to A₂-adenosine receptors in rabbit striatal membranes, in control membranes and after treatment (1-hr preincubation at 25°) with 20 nm (upper) or 100 nm (lower) m-DITC-APEC. The volume of incubation for radioligand binding (approximately 150 μg of protein/tube) was 1 ml. Membranes were incubated with radioligand at 25° for 90 min. Total binding in control (●) and treated ○ membranes is shown. Nonspecific binding in control (×) and treated (Δ) membranes was nearly identical.

Fig. 8

Protection by NECA and theophylline of A_2a-adenosine receptors in rabbit striatal membranes during a 20-min preincubation at 25° with 20 nm ○ or 100 nm 4 concentrations of the affinity label m-DITC-APEC (1), as indicated by binding of [³H]CGS 21680 (5 nm) and [³H]XAC (1 nm).

Fig. 9

Time course for inhibition of rabbit striatal A_2a-adenosine receptors, at 25° by 100 nm p-DITC APEC (4). [³H]CGS 21680 was used at a concentration of 5 nm. This curve is representative of data from four separate experiments.

Fig. 10

Transmembrane model of the canine A₂-adenosine receptor, as proposed by van Galen et al. (figure reproduced from Ref. with permission from authors).