Blockade of granulosa cell differentiation by an antagonistic analog of adenosine 3',5'-cyclic monophosphate (cAMP): central but non-exclusive intermediary role of cAMP in follicle-stimulating hormone action.
The intermediary role and relative importance of cAMP in follicle-stimulating hormone (FSH) hormonal action were reinvestigated at the level of the rat granulosa cell employing Rp-cAMPS, a novel antagonistic analog of cAMP. This approach may not only provide for direct documentation of cAMP dependence, but may also, by inference, highlight the potential relative importance of other putative intracellular second messenger systems. Initial cell-free validation studies indicated that Rp-cAMPS is capable of effectively competing with cAMP for binding to and activation of the regulatory subunit of the granulosa cell A-kinase holoenzyme. Subsequent whole-cell studies employed cultured rat granulosa cells, the cAMP-phosphodiesterase activity of which was suppressed with ZK62711. Basal progesterone accumulation was relatively low, remaining unaffected by treatment with a maximally effective dose of Rp-cAMPS by itself (10(-3) M). Whereas treatment with FSH (30 ng/ml) resulted in a substantial increase in progesterone accumulation, concurrent treatment with increasing concentrations (10(-6)-10(-3) M) or Rp-cAMPS brought about dose-dependent decrements in the FSH effect with a median effective dose of 1.8 +/- (SE) 0.4 x 10(-5) M and a maximal, but incomplete inhibitory effect of 70 +/- (SE) 6%. Higher concentrations of FSH (greater than or equal to 100 ng/ml) progressively diminished, but did not abolish the Rp-cAMPS blockade. Removal of Rp-cAMPS resulted in progressive resumption of FSH responsiveness suggesting reversibility of action. Significantly, Rp-cAMPS proved highly effective in blocking the action of its agonistic diastereomer Sp-cAMPS. However, Rp-cAMPS was unable to block the action of the lactogenic receptor agonist prolactin, the second messenger of which remains uncertain. Taken together, these findings provide additional direct support to the notion that cAMP may be an intracellular second messenger of FSH. However, to the extent that Rp-cAMPS is incapable of complete neutralization of FSH action, our findings further suggest that cAMP may play a central, albeit non-exclusive role in FSH-supported granulosa cell differentiation and that other putative second messenger systems may also be at play.
Adashi
E Y
EY
Department of Obstetrics/Gynecology, University of Maryland School of Medicine, Baltimore 21201.
Resnick
C E
CE
Jastorff
B
B
D000818:Animals; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000166:cytology* / Q000187:drug effects; D011374:Progesterone / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015290:Second Messenger Systems; D013237:Stereoisomerism; D013873:Thionucleotides / Q000494:pharmacology*
D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D011374:Progesterone; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D011494:Protein Kinases