title: Inhibition of luteinizing hormone/human chorionic gonadotropin-stimulable adenylyl cyclase activity in rat luteal membranes by nonsteroidal component(s) in human follicular fluid.
abstract: We examined the ability of nonsteroidal components of human follicular fluid (hFF) to alter gonadotropin responsiveness using the LH/hCG-sensitive adenylyl cyclase system of rat luteal membranes. Follicular aspirates were obtained from regularly ovulatory women (n = 10) whose follicles were stimulated by human menopausal gonadotropin and hCG as part of an in vitro fertilization program. hFF from large follicles was pooled and extracted with 10% (wt/vol) activated charcoal. Maximal hCG stimulation of adenylyl cyclase activity obtained with 10 micrograms/ml hCG and 100 microM of the hydrolysis-resistant GTP analog guanyl 5'-yl-imidodiphosphate was significantly inhibited by hFF in a dose-dependent manner. Addition of about 500 micrograms hFF protein caused inhibition of 70% compared to the control value. Fractionations of hFF by ultrafiltration using membranes of precalibrated pore size demonstrated that the inhibitory activity was associated with a less than 10,000 mol wt fraction; 3 micrograms protein/assay of this fraction resulted in 50% inhibition (IC50) of maximal hCG stimulation. The inhibitory activity also passed through an Amicon YM-2 membrane (mol wt retention, 1,000), but not through an Amicon YC-05 membrane (mol wt retention, 500). An IC50 of about 0.01 microgram protein/assay was found for both the 500-1,000 and the 1,000-5,000 mol wt fractions. NaF or forskolin-stimulated adenylyl cyclase activity was not altered by unfractionated hFF or by the 500-10,000 mol wt subfractions, suggesting that inhibition was limited to LH/hCG stimulation. Further analysis of the effects of low mol wt fraction on hCG stimulation of adenylyl cyclase indicated that enzyme inhibition was not accompanied by a shift in the hCG concentration required for half-maximal stimulation (the apparent activation constant) compared to dose-response curves obtained in the absence of added fraction. Equilibrium binding studies showed that [125I]hCG interaction with luteal membranes was significantly inhibited by hFF; 7 micrograms protein/assay of the less than 10,000 mol wt fraction reduced specific binding by 60%. Moreover, kinetic analysis carried out in the absence or presence of a fixed amount of low mol wt fractions revealed a competitive type of binding inhibition. Our data demonstrate that a nonsteroidal component(s) of hFF has a direct inhibitory effect on LH/hCG-responsive luteal adenylyl cyclase and that the inhibitor(s) exerts its actions through a mechanism involving competition with LH/hCG for the same binding sites.
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001826:Body Fluids / Q000032:analysis / Q000502:physiology; D006063:Chorionic Gonadotropin / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003338:Corpus Luteum / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007986:Luteinizing Hormone / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008970:Molecular Weight; D006080:Ovarian Follicle / Q000032:analysis / Q000502:physiology*; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology; D014462:Ultrafiltration
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D006063:Chorionic Gonadotropin; D005576:Colforsin; D012969:Sodium Fluoride; D007986:Luteinizing Hormone; D000262:Adenylyl Cyclases