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1.
Steroids ; 71(9): 785-91, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16797624

ABSTRACT

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. In vitro studies to determine the mechanism of action are hindered because OTR spontaneously upregulates in vitro and it is impossible to alter expression with P4 or estradiol. During recent studies examining the effect of P4 and an antagonist (mifepristone) on PG secretion, we found that mifepristone attenuated OT-stimulated PG secretion from endometrial epithelial cells. The objective of the present study was to determine, whether this effect of mifepristone was due to changes in prostaglandin synthesis and/or OTR. A time-course showed that mifepristone (5 microM) had no significant effect after 24 h but by 72 h it decreased PGF(2alpha) secretion (P<0.01) and abolished the response of the cells to OT (P<0.01). The presence or absence of P4 did not affect the response to mifepristone. To determine the site of action of mifepristone, cells were cultured for 72 h with or without mifepristone and then COX-1 and COX-2 were measured by Western blotting and OTR was measured by saturation analysis. The results showed that mifepristone did not affect basal or PMA-stimulated expression of either COX-1 or COX-2 but did, however, decrease OTR number (P<0.05). These data demonstrate that OTR and the response to OT can be downregulated in endometrial epithelial cells in vitro via a mechanism involving the P4 receptor.


Subject(s)
Dinoprost/biosynthesis , Endometrium/cytology , Endometrium/drug effects , Progesterone/antagonists & inhibitors , Receptors, Oxytocin/antagonists & inhibitors , Animals , Cattle , Cells, Cultured , Cyclooxygenase 1/biosynthesis , Cyclooxygenase 2/biosynthesis , Endometrium/metabolism , Estrous Cycle/metabolism , Female , Gene Expression Regulation/drug effects , Mifepristone/pharmacology
2.
Am J Vet Res ; 66(11): 1985-91, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16334960

ABSTRACT

OBJECTIVE: To characterize expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and regulation of prostaglandin E2 (PGE2) production by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage from the metacarpophalangeal joints of 7 adult horses. PROCEDURE: Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin-1beta (rhIL-1beta) for 24 hours and then with rhIL-1beta (5 ng/mL) for 3, 6, 9, 12, and 24 hours. Concentration of PGE2 in the media was measured via radioimmunoassay. Total RNA was extracted from harvested chondrocytes, and regulation of COX-2 and mPGES-1 mRNA was studied via reverse transcriptase-polymerase chain reaction assay and Southern blot analysis with equine-specific probes. Western blot analyses were performed on cellular extracts to characterize expression of COX-2 and mPGES-1 protein. RESULTS: Stimulation with 5, 10, and 20 ng of rhIL-1beta/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1beta (5 ng/mL) for 6 to 24 hours increased PGE2 production significantly. The increase in prostaglandin production was associated with an induction of COX-2 and mPGES-1 transcripts. There also was an rhIL-1beta-dependent induction in COX-2 and mPGES-1 protein expression. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, results indicated that the rhIL-1beta-dependent increase in PGE2 production in equine chondrocytes in monolayer culture was associated with coordinated upregulation of COX-2 and mPGES-1 expression. The pathophysiologic consequences of upregulated COX-2 and mPGES-1 expression and of PGE2 synthesis in rhIL-1beta-stimulated equine chondrocytes remain to be elucidated.


Subject(s)
Cartilage, Articular/enzymology , Cyclooxygenase 2/biosynthesis , Horses/metabolism , Interleukin-1/pharmacology , Intramolecular Oxidoreductases/biosynthesis , Animals , Blotting, Western/veterinary , Cartilage, Articular/drug effects , Chondrocytes/drug effects , Chondrocytes/enzymology , Cyclooxygenase 2/genetics , Dinoprostone/biosynthesis , Enzyme Induction/drug effects , Intramolecular Oxidoreductases/genetics , Prostaglandin-E Synthases , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary
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