Molecular identification of adenylyl cyclase 3 in bovine corpus luteum andits regulation by prostaglandin F2 alpha-induced signaling pathways

The involvement of cAMP in various aspects of ovarian steroidogenic cells f unctions has been extensively studied. However, the adenylyl cyclase (AC) t ypes expressed in ovarian cells, of any species, are not yet determined. Th e present study was undertaken to identify AC types present in bovine lutea l cells and their regulation by various stimuli. AC isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain by Northern blotting analysis, whereas the bovine corpus luteum (CL) only expressed AC3 and 6 mRNAs, with AC3 being more abundant than AC6. The use of AC3-specific primers in RT-PCR reaction verified the presence of AC3 mRNA in both bovine and rat CL tissu e as well as in bovine steroidogenic luteal cells. Because these two AC iso forms, AC3 and 6, exhibit distinct regulatory patterns we have next examine d the effects of various signaling pathways on AC activity in luteal cells. These studies have shown that: 1) prostaglandin (PG) F2 alpha and phorbol 12-myristate 13-acetate markedly elevated agonist-stimulated cAMP synthesis (these effects were inhibited by addition of highly specific PKC inhibitor , bisindolylmaleimide); 2) depletion of Ca2+ from the incubation medium inh ibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 m M) significantly stimulated cAMP production in luteal cells; and 4) the eff ects of Ca2+ on cAMP synthesis were evident only in the presence of forskol in. These regulatory characteristics of AC activity are consistent with the molecular identification of ACs indicating the presence of AC3 in luteal c ells. The reported data may delineate the cross-talk between physiological activa tors of AC in the CL (such as LH, PGE(2), and PGI(2)) and other ligands (su ch as PGF2 alpha and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward understanding the mode of action of a wide array of h ormones regulating ovarian cells.