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

Abstract

1. 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. 2. 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 (K(D)s) of 5.66±0.44 nm (n = 4). 3. Specific [3H]-BWA868C bound to DP receptors with a high affinity (K(D) = 1.45±0.01 nM, n = 3) and to a finite, saturable number of binding sites (B(max) = 21.1±0.6 nmol g-1 wet weight). 4. 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. 5. 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. 6. 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.