title: Adenosine 3',5'-monophosphate-dependent protein kinase and granulosa cell responsiveness to gonadotropins.
abstract: The mechanisms by which estradiol enhances the actions of FSH (and cAMP), including the induction of LH receptors in rat ovarian granulosa cells, remain unclear. These studies were conducted to determine the extent to which changes in the activity, content, or intracellular distribution of the catalytic subunit of cAMP-dependent protein kinase might be altered in granulosa cells as a consequence of estradiol, FSH, and hCG administration in vivo. Dose-dependent stimulation of protein kinase activity (measured by histone phosphorylation in the presence of [gamma 32P]ATP and cAMP) demonstrated that the EC50 for cAMP was consistently 20 X 10(-8) M in cytosols prepared from granulosa cells of hypophysectomized rats before and after treatment with estradiol alone or estradiol and FSH. However, estradiol alone caused a 1.5 to 2.0-fold increase in the total amount of enzyme activity. When the cytosol content of the catalytic subunit (C) was quantitated directly, using immunoblotting procedures, the amount of C was 40 pmol/mg protein in all tissues, regardless of hormone treatments in vivo. When the content of RII, the regulatory subunit of type II cAMP-dependent protein kinase, was measured by similar immunoblotting procedures, a 10-fold increase was observed in granulosa cells exposed to both estradiol and FSH compared to that in cells exposed to estradiol alone. Greater than 80% of the intracellular content of both C and RII was present in the cytosol fraction (30,000 X g supernatant) rather than in the particulate nuclear fraction (30,000 X g pellet) of granulosa cells. This distribution of subunits was not altered by rapidly elevating intracellular concentrations of cAMP in vivo with 10 IU hCG, iv. We conclude that the catalytic subunit of protein kinase is a constitutive component of granulosa cells and that the sensitivity of the enzyme for cAMP is not affected by hormones or by a 10-fold increase in RII. Thus, the ability of estradiol to enhance FSH and cAMP action in granulosa cells appears to come primarily from the induction of specific substrates for the enzyme and a small increase in the catalytic activity but not from a change in the content of the catalytic subunit.
mesh_terms: D000818:Animals; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D007016:Hypophysectomy; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D006063:Chorionic Gonadotropin; D046911:Macromolecular Substances; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase