Oxidized-low density lipoprotein inhibits cyclic AMP production by porcine luteal cells.
Oxidized(OX)-low density lipoprotein (LDL) inhibits steroidogenesis by luteal cells (LC) from regressing porcine CL. The present study was designed to investigate the mechanism of inhibition by determining whether OX-LDL inhibits basal and agonist-stimulated cAMP production in regressing LC. Collagenase-dispersed porcine LC (n = 7 animals, estrous cycle Day 12-15) were cultured (2.5 x 10(5) cells/0.5 ml) in serum-free DMEM/Hams F-12 in duplicate wells at 37 degrees C. Approximately 18 hr after plating, media were replaced and LC were immediately treated with human LDL (0, 25, or 100 microg/ml) or OX-LDL (25 or 100 microg/ml). LC were incubated for 2 hr before addition of isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity, immediately followed by hCG (100 ng/ml), cholera toxin (CT; 0.1 microM), forskolin (FS; 50 microM), or no further treatment (controls). LC were incubated for an additional 90 min. After removal of culture media, cells were extracted with 0.1 N HCl. Cell extracts were assayed for cAMP by enzyme immunoassay (EIA). HCG, CT, and FS increased (P < 0.05) cAMP production approximately four-, 10-, and 25-fold, respectively, relative to controls. OX-LDL (25 and 100 microg/ml) inhibited (P < 0.05) cAMP production by unstimulated, hCG-, and CT-stimulated LC, but not that by FS-stimulated LC. The highest concentration of OX-LDL (100 microg/ml) reduced cAMP formation by 39.8 +/- 6.6%, 44.7 +/- 10.5%, and 67.7 +/- 4.5% in unstimulated, hCG-, and CT-stimulated LC, respectively. In contrast, unmodified LDL (25 and 100 microg/ml) did not alter cAMP production. We conclude that OX-LDL can interfere with the cAMP signaling pathway in regressing luteal cells by acting at sites proximal to adenylate cyclase activation.
Brannian
J D
JD
Department of OB/GYN, University of South Dakota School of Medicine, Sioux Falls 57105, USA.
Rickert
C S
CS
D015056:1-Methyl-3-isobutylxanthine / Q000737:chemistry; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D002772:Cholera Toxin / Q000494:pharmacology; D006063:Chorionic Gonadotropin / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis / Q000096:biosynthesis*; D005260:Female; D007124:Immunoenzyme Techniques / Q000662:veterinary; D008077:Lipoproteins, LDL / Q000502:physiology*; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D013552:Swine / Q000502:physiology*
D000276:Adjuvants, Immunologic; D006063:Chorionic Gonadotropin; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D005576:Colforsin; D002772:Cholera Toxin; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine