PROSTACYCLIN ACTIVATION OF ADENYLYL CYCLASE IN RABBIT CORPUS-LUTEUM MEMBRANES - COMPARISON WITH 6-KETO PROSTAGLANDIN F1-ALPHA AND PROSTAGLANDIN-E1

Rabbit corpus luteum membranes contain an adenylyl 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 concentration 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 condition, 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 activities were dependent on Mg ion (MgCl2 added in excess of ATP plus EDTA). In the absence of guanyl nucleotide, half-maximal activates 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 activites are not additive. Futher 13 - 40-fold excess 6-keto PGF(1α) does not alter PGI2 or PGE1 stimulation of adenylyl cyclase.