Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes -: a new, selective antagonist radioligand for A2A adenosine receptors

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [H-3]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([H-3]MSX-2). [H-3]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [H-3]MSX-2 labeled a single class of binding sites with high affinity (K-d = 8.0 nM) and limited capacity (B-max = 1.16 fmol . mg(-1) of protein). The presence of 100 mu M GTP, or 10 mM magnesium chloride, respectively, had no effect on [H-3]MSX-2 binding. AR agonists competed with the binding of 1 nM [H-3]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)> 2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) >2-chloroadenosine (2-CADO) >N-6-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)> (R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K-i values for antagonists were in accordance with data from binding studies with the agonist radioligand [H-3]CGS21680, while agonist affinities were 3-7-fold lower, [H-3]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM. (C) 2000 Elsevier Science B.V. All rights reserved.