Interaction of prostanoid EP3 and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE2.

BACKGROUND AND PURPOSE: Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E2 (DX-DM PGE2) on pig cerebral artery when used as a selective EP3 receptor agonist. This study investigated the selectivity profile of DX-DM PGE2, focusing on the interaction between its EP3 and TP (thromboxane A2-like) agonist activities. EXPERIMENTAL APPROACH: Contraction of guinea-pig trachea (EP1 system) and aorta (EP3 and TP systems) was measured in conventional organ baths. KEY RESULTS: Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE2 (EP3 agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE2 induced strong contraction, which on the basis of treatment with (DG)-3ap (EP3 antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP3 and TP receptors. EP3/TP self-synergism also accounted for contraction induced by PGF(2α) and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE2 also showed significant EP1 agonism on guinea-pig trachea as defined by the EP1 antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM. CONCLUSIONS AND IMPLICATIONS: EP3/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP3 agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP3 system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed.

Cloning of a carboxyl-terminal isoform of the prostanoid FP receptor.

An FP prostanoid receptor isoform, which appears to arise from alternative mRNA splicing, has been cloned from a mid-cycle ovine large cell corpus luteum library. The isoform, named the FP(B) receptor, is identical to the original isoform, the FP(A), throughout the seven transmembrane domains, but diverges nine amino acids into the carboxyl terminus. In contrast to FP(A), whose carboxyl terminus continues for another 46 amino acids beyond the nine shared residues, the FP(B) terminates after only one amino acid. The FP(A) isoform appears to arise by the failure to utilize a potential splice site, while a 3.2-kilobase pair intron is spliced out from the FP gene to generate the FP(B) isoform mRNA. The two isoforms have indistinguishable radioligand binding properties, but seem to differ in functional coupling to phosphatidylinositol hydrolysis. Thus, in COS-7 cells transiently transfected with either the FP(A) or the FP(B) receptor cDNAs, prostaglandin F(2alpha) stimulates inositol phosphate accumulation to the same absolute maximum, but the basal level of inositol phosphate accumulation is approximately 1.3-fold higher in cells transfected with the FP(B) as compared with cells transfected with the FP(A) isoform. Using the polymerase chain reaction, mRNA encoding the FP(B) isoform was identified in the ovine corpus luteum.

Cloning of a receptor for prostaglandin F2 alpha from the ovine corpus luteum.

A complementary DNA clone encoding a functional receptor for prostaglandin F2 alpha (PGF2 alpha) has been isolated from an ovine large luteal cell complementary DNA library (prepared from day 10 mid-luteal phase RNA). This receptor, which has been designated FP, consists of 362 amino acids (M(r) = 40,982) and is a member of the family of G protein-coupled receptors. Radioligand binding studies with membranes prepared from transfected COS cells demonstrated specific 17-[3H]phenyl-trinor-PGF2 alpha binding that was displaced by prostanoids in the order of 17-phenyl-trinor-PGF2 alpha > PGF2 alpha > fluprostenol > PGD2 > PGE2 >> 8-epi PGF2 alpha. Xenopus laevis oocytes injected with RNA encoding the ovine FP receptor responded to 17-phenyl-trinor-PGF2 alpha with increased membrane chloride conductance in calcium-free medium. Northern blot analysis with RNA from day 10 corpus luteum showed a major band of approximately 6.1 kilobases. On day 14, when luteolysis usually starts, the abundance of this 6.1-kilobase band was variable between individual ewes, and on day 16, when luteolysis is underway, the message was uniformly less abundant. This variability appeared to correlate with circulating progesterone. Thus, when the progesterone level was high (days 10 and 14 depending on whether luteolysis had started), the amount of FP receptor message was high, whereas when the progesterone level was low or falling (day 16), the amount of FP receptor message decreased. We have cloned an ovine FP receptor whose expression confers appropriate functional activity in COS cells and Xenopus oocytes. Furthermore, the level of messenger RNA encoding the FP receptor is high in the midluteal phase ovine corpus luteum and decreases during luteolysis.