TRIFUNCTIONAL LIGANDS: A RADIOIODINATED HIGH AFFINITY ACYLATING ANTAGONIST FOR THE A1 ADENOSINE RECEPTOR

Abstract

A new xanthine (adenosine antagonist) radioligand that binds covalently to A_1-adenosine receptors was prepared and used as a receptor probe. BH-DITC-XAC was synthesized via a trifunctional aryl diisothiocyanate crosslinker. containing the p-hydroxyphenylpropionyl group for radioiodination. The xanthine competed against agonist or antagonist A₁ receptor radioligands in bovine brain membranes with an IC₅₀, of 40nM. ¹²⁵I-BH-DITC-XAC, prepared directly by the chloramine T method and purified by HPLC. bound specifically to A₁ receptors. This binding was inhibited in the presence of the adenosine agonists R-PIA, S-PIA. and NECA in a dose dependent manner and with the order of potency characteristic of bovine A₁ receptors. Incubation of affinity purified bovine A₁-receptors with ¹²⁵I-BH-DITC-XAC (0.8 nM) for 2 hours resulted in the specific and clean labelling of a polypeptide band corresponding to MW 36,000, identical to that previously found for the A₁ receptor.

Keywords: adenosine receptors; affinity labeling; radioiodination; xanthines.

FIGURE 1

Synthesis of trifunctional XAC derivatives that act as irreversible antagonists at A₁ adenosine receptors.

FIGURE 2

Synthesis of non-radioactive xanthine standards and intermediates.

FIGURE 3

Inhibition of binding of [¹²⁵I]APNEA to bovine cerebral cortical A₁-adenosine receptors by the trifunctional xanthine BH-DITC-XAC, 3b, a substrate for radioiodination. The binding was carried out at 37°C for 60 min. [¹²⁵I]APNEA was present at a concentration of 0.5 nM in 0.05M TRIS at pH 8.26. 10mM MgCl₂, 1 mM EDTA, at 5°C. Nonspecific binding was defined with 10⁻⁵ M R-PIA.

FIGURE 4

Inhibition of binding of ¹²⁵I-BH-DITC-XAC to bovine cerebral cortical A₁-adenosine receptors by adenosine agonists: R-PIA (○), S-PIA (□), and NECA (△). The binding was carried out at 25°C for 90 min. ¹²⁵I-BH-DITC-XAC was present at a concentration of 0.8 nM in 0.05 M TRIS buffer at pH 7.4. Nonspecific binding was defined with 10⁻⁵ M R-PIA.

FIGURE 5

Affinity labeling of A₁-adenosine receptors purified from bovine brain. Membranes were incubated with the antagonist affinity probe ¹²⁵I-BH-DITC-XAC (0.8 nM) at pH 7.4 for two hours in the absence and presence of 5 mM theophylline. The labeled receptor preparation was then subjected to SDS-PAGE and autoradiography. Molecular weight standards are shown on the left.