Human gel-filtered platelets (GFP) and radiolabeled prostacyclin (PGI2), prostaglandin (PG) E2 and PGE1, were used to ascertain whether PGI2 and PGs of the E series share a common receptor or have their own specific receptors on platelets. Attention was given to ensuring the proper experimental conditions to compensate for the rapid half-life of PGI2 at physiologic pH. Specific [3H]PGI2 binding to GFP was maximal at 5 min and pH 7.45. Scatchard analysis indicated a single class of binding sites with an apparent K(D) of 4.52 x 10-8 M and 1130 sites per platelet. Approximately 90% of specifically bound [3H]PGI2 could be dissociated by excess unlabeled PGI2 by 5 min. The IC50 for PGI2 was 66 nM. By 5 min, PGE1 and PGE2 were only 7.17 and 0.03%, respectively, as potent inhibitors of binding. Maximal specific binding of either [3H]PGE2 or [3H]PGE1 to GFP occurred by 60 min. During 60-min incubations with [3H]PGE2, the IC50 values for PGE2 and PGE1 were 3 and 6 nM, respectively. When [3H]PGE1 was used, the IC50 values for PGE1 and PGE2 were 30 and 10 nM, respectively. To examine PGI2 competition for [3H]PGE2 and [3H]PGE1 binding sites, 5-min incubation periods were used. PGI2 was only 0.38% as potent an inhibitor of [3H]PGE2 compared to PGE2 and only 30% as potent an inhibitor of [3H]PGE1 compared to PGE1. Scatchard analysis of the 60-min competition experiments using [3H]PGE2 and [3H]PGE1 and the homologous unlabeled ligand yielded curvilinear plots in both instances. However, when GFP were preincubated with a saturating concentration of PGE2, only low affinity/high density binding sites were available for subsequent [3H]PGE1 binding. In contrast, this maneuver did not affect subsequent [3H]PGI2 binding. Consequently, high affinity binding [3H]PGE1 appears to be fully accounted for by binding of this ligand to PGE2 sites. The authors interpret these data to indicate that there are separate and distinguishable receptors for PGI2 and PGE2 on human GFP because 1) PGI2 competes rather poorly for either [3H]PGE2 or [3H]PGE1 binding; 2) neither PGE2 nor PGE1 compete well for [3H]PGI2 binding; 3) preincubation with a saturating concentration of PGE2 before [3H]PGI2 binding does not influence [3H]PGI2 binding; and 4) the concentration of PGE2 required to complete for [3H]PGI2 binding is approximately 1000 times greater than the concentration required to saturate fully all binding sites occupied by [3H]PGE2. There is no evidence for a high affinity PGE1-specific receptor because all PGE1 binding can be accounted for by interactions with the PGE2 sites.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1986|