Prostaglandin DP receptors positively coupled to adenylyl cyclase in embryonic bovine tracheal (EBTr) cells: pharmacological characterization using agonists and antagonists

Abstract

1 Various prostaglandin agonists representing various classes of receptor subtypes were evaluated for their ability to stimulate adenylyl cyclase via the endogenous DP receptor in embryonic bovine tracheal (EBTr) cells. Two antagonists were used to block the agonist-induced cyclic AMP production. 2 ZK118182 (EC 50 =16+4 nM), RS-93520 (EC 50 =23+4 nM), SQ27986 (EC 50 =33+9 nM), ZK110841 (EC 50 =33+5 nM), BW245C (EC 50 =59+19 nM) and PGD 2 (EC 50 =101+10 nM) (n=4 ± 70) were the most potent agonists. Whilst most compounds were full agonists (E max =100% relative to PGD 2 ), BW245C was signi®cantly more e cacious than PGD 2 (E max =121+3%; P50.001) and RS-93520 appeared to be a partial agonist (E max =64+9%; P50.001).3 Agonists from the EP (e.g. enprostil; misoprostol; butaprost), FP (e.g. cloprostenol;¯uprostenol; PHXA85), IP (iloprost; PGI 2 ) and TP (U46619) prostanoid receptor classes were weak agonists or inactive in the EBTr cell assay system. 4 The DP-receptor antagonist, BWA868C, showed a competitive antagonist pro®le with pA 2 values of 8.00+0.02 and 8.14+0.13 in Schild analyses with two structurally di erent agonists, BW245C and ZK118182, respectively (n=3). AH6809, another purported DP-receptor antagonist, weakly inhibited PGD 2 -and ZK118182-induced cyclic AMP production (K i s=808+193 nM and 782+178 nM, respectively). 5 The current studies have characterized the DP receptor positively coupled to adenylyl cyclase in EBTr cells using a wide range of agonist and antagonist prostaglandins. These data support the utility of the EBTr cell line as a useful tool for the evaluation of DP receptor agonists and antagonists and for pro®ling other classes of prostaglandins. Keywords: Prostaglandin; DP receptor; EBTr cells; BW245C; BWA868C; AH6809; ZK118182; RS93520; cyclic AMP; adenylyl cyclaseAbbreviations: EC 50 , concentration of agonist required to produce 50% of the maximal response (potency); E max , per cent of the maximal response (intrinsic activity; e cacy); IC 50 , concentration of inhibitor required to produce 50% of the maximal inhibition; K i , concentration of inhibitor required to produce 50% of the maximal inhibition at equilibrium; pA 2 , equilibrium dissociation constant of the antagonist; RIA, radioimmunoassay

A Reporter Gene Assay for High-Throughput Screening of G-Protein-Coupled Receptors Stably or Transiently Expressed in HEK293 EBNA Cells Grown in Suspension Culture

“…10A). This antagonism of AH6809 at the DP receptor has also been reported by others (16,17). At the highest concentration (10 M) tested, AH6809 induced a slight but reproducible increase (25%) in the maximal SEAP activity attained at the EP 1 receptor with iloprost concentrations of 300 nM and above.…”

“…The calculated pK B value for AH6809 on the DP receptor was 6.7 (K B of 180 nM) (Fig. 10C), which is fourfold lower than the recently reported K i value of 800 nM (17). robust, flexible, and amenable to high-throughput screening.…”

International Union of Basic and Clinical Pharmacology. LXXXIII: Classification of Prostanoid Receptors, Updating 15 Years of Progress

“…Other potent DP 1 agonists containing a 15-cyclohexyl group include RS-93520 (Fig. 2), a prostacyclin analog with completely inverted stereochemistry in the bicyclic ring (Alvarez et al, 1991;Crider et al, 1999) and SQ-27986 (Fig. 2), a PGH 2 analog with correspondingly inverted stereochemistry (see Fig.…”

Prostaglandin D2inhibits fibroblast migration

“…The DP receptor is known to couple to adenylyl cyclase and increase cAMP [16], and other agonists are known to inhibit fibroblast chemotaxis through cAMP [17]. To confirm a cAMP-mediated inhibition of fibroblast chemotaxis by PGD 2 , the protein kinase A (PKA) inhibitor KT5720 was used.…”