Characterization of [3H]-N-[R-(2-benzothiazolyl)thio-2-propyl]-2-chloroadenosine ([3H]-NNC 21-0136) binding to rat brain:: Profile of a novel selective agonist for adenosine A1 receptors

N-[R-(2-Benzothiazolyl)thio-2-propyl]-2-chloroadenosine (NNC21-0136) is a novel adenosine agonist with neuroprotectant properties in rodent models of focal and global ischemia that exhibits diminished cardiovascular side effects when compared to reference A(1) agonists [Sheardown et al., 1995]. NNC 21-0136 is shown to be a potent agonist of adenosine A(1) receptors showing around 60- and 30-fold selectivity over adenosine A(2A) and A(3) receptors, respectively. In order to further characterize the central nervous system molecular target for NNC 21-0136, the compound has been radiolabeled and utilized in receptor binding experiments. In vitro receptor autoradiography with [H-3]-NNC 21-0136 revealed high levels of receptors in the CA1 region of the hippocampus, the granule cell layer of the cerebellar cortex, and moderate uniform levels throughout the cerebral cortex. [H-3]-NNC 21-0136 binding to rat cerebral cortical membranes was reversible and saturable (B-max = 0.98 +/- 0.04 pmol/mg protein) to a high affinity site (K-d = 1.16 +/- 0.06 nM) as determined by saturation binding experiments. [H-3]-NNC 21-0136 binding was sensitive to guanosine-5'-O-(3-thio)triphosphate-gamma-S and enhanced by the presence of divalent cations (Ca2+ and Mg2+). A highly significant correlation between the affinities of several compounds to displace [H-3]-NNC 21-0136 binding as compared to [H-3]-N-R-(2-phenylisopropyl)adenosine ([H-3]-R-PIA) binding to adenosine A(1) receptors in rat cortical membranes was observed. We conclude that [H-3]-NNC 21-0136 is a new, selective radioligand for adenosine Al receptors. (C) 1997 Wiley-Liss, Inc.