Characterisation of [3H]-ZM 241385 binding in wildtype and A2A knockout mouse brain   M.D.W. Kelly, *C. Ledent, I. Kitchen & S.M.O. Hourani, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, UK. *IRIBHN, Université Libre de Bruxelles, Bruxelles, Belgium.

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[³H]-ZM 241385 has been shown to be a highly selective, high affinity adenosine A_2A receptor antagonist ligand in the rat brain (Alexander et al., 2001). Adenosine A_2A receptor knockout mice have been generated (Ledent et al., 1997), and provide a unique opportunity to explore the true selectivity of this ligand. In this study we have therefore used [³H]-ZM 241385 to quantify and localise the distribution of adenosine A_2A receptors in the brain and spinal cord of both wild-type (+/+) and A_2A knockout (-/-) mice.

Adult male +/+ CD1 mice (30-35g) and their -/- littermates were obtained from an in-house breeding programme, killed by cervical dislocation and their brains and spinal cords removed. Saturation binding was carried out in triplicate in homogenates of brain (minus cerebellum) for 30 min as described by Alexander et al. (2001). The concentration range of [³H]-ZM 241385 used was 0.25-16nM and non-specific binding was determined in the presence of 5mM theophylline. Saturation analysis was carried out using Graph Pad PRISM. Autoradiographic binding and quantitative analysis were carried out as previously described (Kitchen et al., 1997). Both coronal and sagittal sections (20 µm) cut at 300µm intervals throughout the brain were exposed to 2.5nM [³H]-ZM 241385 for 90 min and non-specific binding was determined using 5mM theophylline (Alexander et al., 2001).

Saturable specific binding was observed in the homogenate binding studies in tissues from +/+ mice, with a B_max of 299.1 ± 28.4 fmolmg^-1 protein and a K_D of 0.75 ± 0.08nM (n=5). Specific binding at K_D was 90-95%. Only a low level of non-saturable binding, 5.0 ± 1.2 fmolmg^-1 protein at K_D (n=4), was observed in -/- brain homogenates (Figure 1).

Figure 1. Specific binding of [³H]-ZM 241385 to brain homogenates from +/+ and -/- mice. Similar results were found in each of at least 4 experiments.

Specific binding of [³H]-ZM 241385 (fmol/mg, n=5, mean ± s.e.m) in +/+ brain slices was confined to the caudate putamen (525.7 ± 69.9), nucleus accumbens (core 369.9 ± 61.2, shell 260.8 ± 25.3), olfactory tubercle (955.1 ± 87.4) and globus pallidus (169.5 ± 44.4). In all other regions of the brain and spinal cord specific binding was either below the limits of detection or <3.0 fmolmg^-1, as was binding in -/- brains.

Overall these results confirm the selectivity of [³H]-ZM 241385 as a radioligand for A_2A receptors, and also confirm the lack of A_2A receptors in the spinal cord and extrastriatal brain regions of the mouse, as previously shown using the agonist radioligand [³H] CGS 21680 (Bailey et al., 2002).

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Ledent C. et al. (1997) Nature 388, 674-678.