A new high affinity, iodinated adenosine receptor antagonist as a radioligand/photoaffinity crosslinking probe

Abstract

A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenylacetylamino)ethyl]amino]carbonyl]- methyl]oxy]phenyl]-1,3-dipropylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD = 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125I]PAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA greater than S-PIA greater than NECA much greater than IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.

Fig. 1

[¹²⁵I]PAPAXAC saturation curves in bovine cerebral cortex membranes. Membranes were prepared as described under Experimental Procedures. Assay volume was 0.25 ml consisting of 150 μl (~100 μg/ml of protein) in 50 mM Tris-HCl, pH 7.4, ~25°, 10 mM MgCI₂, 1 mM EDTA, 50 μl of radioligand, and 50 μl of water or competing ligand (R-PIA 10⁻⁵ m). The incubation was for 2 hr, ~25°. Binding was terminated by vacuum filtration over Schleicher and Schuell glass fiber filters pretreated with 0.3% polyethylenimine for 1 hr, and three washes of 3 ml each with the above buffer with 0.01% CHAPS. ¹²⁵l activity was detected in a Packard gamma-counter at an efficacy of 75%. [¹²⁵I]PAPAXAC was added to a final concentration shown on the abscissa. The data points are means of duplicate determinations. This experiment is representative of four similar experiments. The curves were drawn with the aid of a computer modelling program based on the law of mass action (14).

Fig. 2

Competition of adenosine analogs with [¹²⁵I]PAPAXAC in bovine cerebral cortex membranes. Radioligand binding was performed as described in the legend to Fig. 1 with the indicated concentration of competitors. The lines through the experimentally derived points were drawn with computer-assisted analysis based on a four-parameter logistic equation (13). Radioligand was present at a concentration of 0.3 nM. These curves were replicated twice.

Fig. 3

Photoaffinity crosslinking of [¹²⁵l]PAPAXAC into bovine cerebral cortex membranes. Cerebral cortex membranes were photoaffinity crosslinked with [¹²⁵I]PAPAXAC alone (control) or in the presence of the indicated concentration of competing ligands as described under Experimental Procedures. The samples were then solubilized and electrophoresed on a 12% acrylamide gel. The relative molecular weight (M_r scale) was calibrated using iodinated protein standards: phosphorylase (94,000), bovine serum albumin (67,000), ovalbumin (45,000), carbonic anhydrase (30,000), and soybean trypsin inhibitor (20,000). The results shown are representative of three similar experiments.

Fig. 4

Photoaffinity crosslinking of [¹²⁵I]PAPAXAC into cerebral cortex membranes and [¹²⁵I]AZPNEA labeling of cerebral cortex membranes. Cerebral cortex membranes were prepared as described under Experimental Procedures. Membranes were photoaffinity crosslinked with [¹²⁵l] PAPAXAC and SANPAH either alone (control) or in the presence of R-PIA (10⁻⁶ M) or were directly photoaffinity labeled with [¹²⁵I]AZPNEA either alone or in the presence of 10⁻⁶ m R-PIA as shown on the gel. Samples were then solubilized and electrophoresed on 11% acrylamide gel followed by gel audioradiography. M_r calibration is as described in Fig. 3. This gel is representative of two similar gels.