8-(3-Isothiocyanatostyryl)caffeine Is a Selective, Irreversible Inhibitor of Striatal A(2)-Adenosine Receptors.

8-(3-Isothiocyanatostyryl)caffeine (ISC) was synthesized and shown to inhibit selectively the binding of [(3)H]CGS 21680 (an A(2a)-selective agonist) at adenosine receptors in striatal membranes. The K(i) value at A(2a)-receptors was found to be 110 nM (rat), with selectivity ratios for A(2a) versus A(1)-receptors in rat, guinea pig, bovine, and rabbit striatum of >100-fold. Preincubation of membranes with ISC caused a dose-dependent, irreversible antagonism of the binding of [(3)H]CGS 21680, with an IC(50) value of 3 μM. The irreversibility is likely due to the presence of the chemically reactive isothiocyanate group, since the binding of the corresponding analogue in which the isothiocyanate was replaced with a chloro group was completely reversible. The potency of ISC to irreversibly inhibit the binding of [(3)H]CGS 21680 in several species varied in the order rat ≈ guinea pig > bovine ≈ rabbit. In all four species, binding of the A(1)-selective agonist [(3)H]R-N(6)-phenylisopropyladenosine was not diminished by pre-treatment with 2 μM ISC. The kinetics of irreversible inhibition of rat A(2a)-receptors by 2 μM ISC gave a t(1/2) of approximately 3 min. Following partial inactivation, the remaining rat A(2a)-binding sites retained the same K(d) value as in control membranes for saturation by [(3)H]CGS 21680. Thus, ISC appears to be a selective affinity label for A(2a)- versus A(1)-receptors in the brain.