[125I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

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Abstract
GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]‐GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]‐GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]‐GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]‐GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]‐GR231118/BIBO3304‐insensitive binding sites were detected in few areas. These [125I]‐GR231118/BIBO3304‐insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]‐GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]‐GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. British Journal of Pharmacology (2000) 129, 37–46; doi:10.1038/sj.bjp.0702983