Prostacyclin Activation of Adenylyl Cyclase in Rabbit Corpus Luteum Membranes: Comparison with 6-Keto Prostaglandin F1α and Prostaglandin E11

Abstract

Rabbit corpus luteum membranes contain an adenylyl cyclase system that in addition to being stimulated by luteinizing hormone (LH), is stimulated by both prostacyclin (PGI₂) and prostaglandin E₁ (PGE₁). Stimulation of rabbit corpus luteum adenylyl cyclase by PGI₂, and PGE₁ demonstrates an absolute requirement for guanyl nucleotides. In the presence of 10 µM GTP, 10–20 µg/ml PGI₂ and PGE₁ produce a 3.5–4-fold stimulation of adenylyl cyclase. Under identical conditions, 100 µg/ml 6-keto prostaglandin F_1α (6-keto PGF_1α), the breakdown product of PGI₂, produce only a marginal 1.3-fold stimulation of adenylyl cyclase activity. The concentration at which PGI₂ and PGE₁ stimulate rabbit corpus luteum adenylyl cyclase half-maximally is dependent upon the concentration and type of guanyl nucleotide present at the time of assay. In the presence of low (0.2 µM) GTP, the concentrations of PGI₂ and PGE₁ required for 50% activation were 0.5 and 0.3 µg/ml, respectively. With high (10 µM) GTP in the assay, 1.5 and 2.2 µg/ml PGI₂ and PGE₁, respectively, were required. With 10 µM guanylyl 5′-imidodiphosphate (GMP-P (NH)P, a nonhydrolyzable analog of GTP), on the other hand, concentrations required for half-maximal stimulation differed for each prostanoid, being 0.2 µg/ml for PGI₂ and 0.7 µg/ml for PGE₁. During a standard 0–10 min assay in the absence of prostanoid, GTP and GMP-P(NH)P half-maximally activate luteal adenylyl cyclase at 0.36 and 0.55 µM, respectively. After a 30 min preincubation under adenylyl cyclase assay conditions, GTP and GMP-P(NH)P activated half-maximally at 0.15 and 0.18 µM, respectively. These values remained unaltered upon addition of either PGI₂ or PGE₁. Adenylyl cyclase activites were dependent on Mg ion (MgCl₂ added in excess of ATP plus EDTA). In the absence of guanyl nucleotide, half-maximal activities were obtained at 12 mM Mg ion. After addition of 10 µM of either GTP or GMP-P(NH)P, this value decreased to 3.6 and 1.1 mM, respectively. We found that PGI₂ and PGE₁ had no effect on the Mg ion requirements of the system. It appears that PGI₂ and PGE₁ are acting via the same receptors because their activities are not additive. Further 13–40-fold excess 6-keto PGF_1α does not alter PGI₂ or PGE₁ stimulation of adenylyl cyclase.