PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated p
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BioMed Central
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PKCepsilon and an increase in intracellular calcium concentration are necessary for PGF2alpha to inhibit LH-stimulated progesterone secretion in cultured bovine steroidogenic luteal cells Madhusudan P Goravanahally1, Aritro Sen1,2, Emmet K Inskeep3 and Jorge A Flores*1 Address: 1Department of Biology, West Virginia University, Morgantown, West Virginia, USA, 2Department of Animal Sciences, Michigan State University, East Lansing, Michigan, USA and 3Animal and Veterinary Sciences, West Virginia University, Morgantown, West Virginia, USA Email: Madhusudan P Goravanahally - [email protected]; Aritro Sen - [email protected]; Emmet K Inskeep - [email protected]; Jorge A Flores* - [email protected] * Corresponding author
Published: 30 August 2007 Reproductive Biology and Endocrinology 2007, 5:37
doi:10.1186/1477-7827-5-37
Received: 21 June 2007 Accepted: 30 August 2007
This article is available from: http://www.rbej.com/content/5/1/37 © 2007 Goravanahally et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha 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 PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon 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 PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dosedependent 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 PGF2alpha 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. PKCepsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKCepsilon expression was ablated by 75%, the inhibitory effect of PGF2alpha 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 PKCepsilon 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
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