Pharmacology and autoradiography of human DP prostanoid receptors using [3H]‐BWA868C, a DP receptor‐selective antagonist radioligand

A potent and highly selective DP prostanoid receptor antagonist radioligand, [3H]‐cyclohexyl‐N‐BWA868C (3‐benzyl‐5‐(6‐carboxyhexyl)‐1‐(2‐cyclohexyl‐2‐hydroxyethyl‐amino) hydantoin, ([3H]‐BWA868C)), has been generated for receptor binding and autoradiographic studies. Specific [3H]‐BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23°C and constituted up to 95% of the total binding. The association (K+1) and dissociation (K−1) rate constants of binding were 0.758±0.064 min−1, mmol and 0.0042±0.0002 min−1, respectively, yielding dissociation constants (KDs) of 5.66±0.44 nM (n=4). Specific [3H]‐BWA868C bound to DP receptors with a high affinity (KD=1.45±0.01 nM, n=3) and to a finite, saturable number of binding sites (Bmax=21.1±0.6 nmol g−1 wet weight). DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD2) exhibited high (nanomolar) affinities for [3H]‐BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [3H]‐BWA868C. While [3H]‐PGD2 yielded similar quantitative distribution of DP receptors as [3H]‐BWA868C, the level of non‐specific binding observed with [3H]‐PGD2 was significantly greater than that observed with [3H]‐BWA868C. It is concluded that [3H]‐BWA868C is a high‐affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [3H]‐BWA868C may prove useful for future homogenate‐based and autoradiographic studies on the DP receptor. British Journal of Pharmacology (2000) 131, 1025–1038; doi:10.1038/sj.bjp.0703686