PKC epsilon and an increase in intracellular calcium concentration are necessary for PGF2 alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells.
The hypotheses that PKC epsilon is necessary for: 1) PGF2 alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKC epsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKC epsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2 alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2 alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.PKC epsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKC epsilon expression was ablated by 75%, the inhibitory effect of PGF2 alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKC epsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKC epsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2 alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 mumol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2 alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKC epsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2 alpha.
Goravanahally
Madhusudan P
MP
Department of Biology, West Virginia University, Morgantown, West Virginia, USA. mgoravan@mix.wvu.edu
Sen
Aritro
A
Inskeep
Emmet K
EK
Flores
Jorge A
JA
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D015237:Dinoprost / Q000378:metabolism / Q000494:pharmacology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D007476:Ionophores / Q000494:pharmacology; D008184:Luteal Cells / Q000187:drug effects* / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D011374:Progesterone / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D002614:Chelating Agents; D007476:Ionophores; D034741:RNA, Small Interfering; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000001:Calcimycin; D011374:Progesterone; D004533:Egtazic Acid; D007986:Luteinizing Hormone; D015237:Dinoprost; D051744:Protein Kinase C-epsilon; D002118:Calcium