Submission Number: AYD-4-35-33

Abstract Number: 390

PKC INDUCTION OF PGF SECRETION IS DEPENDENT UPON PROTEIN SYNTHESIS AND REGULATED BY BIFN-.

Aydin Guzeloglu* ¹, Mario Binelli* ¹, Lokenga Badinga* ¹, Thomas R Hansen* ² and William W Thatcher* ¹

Department of Dairy and Poultry Sciences, University of Florida, Gainesville, Florida ¹
Department of Animal Science, University of Wyoming, Laramie, Wyoming ²

Abstract:
Regression of the CL is inhibited by trophoblastic interferon- (bIFN-) at the time of pregnancy recognition in cattle. Phorbol ester (PdBu), an activator of Protein Kinase C (PKC), stimulates prostaglandin F₂ (PGF) in bovine endometrial (BEND) cells. Upon stimulation, PKC translocates from the cytoplasm to cell membrane and is activated. The first objective was to determine if bIFN- inhibits translocation of PKC. Whole cell extracts of BEND cells were analyzed, and , MISSING CHARACTER ENTITY: epsi, and isoforms of PKC identified by Western blotting. Cells were treated with medium alone (control), PdBu (100 ng/ml) or PdBu + bIFN- (50 ng/ml) for 3 and 6h. PdBu induced secretion of PGF (P < 0.01) and bIFN- suppressed (P < 0.01) this secretion by 75% and 86% at 3 and 6 h, respectively. At 3 and 6h, cytosolic and membrane fractions were isolated from cell lysates. Western blotting revealed that all four isoforms of PKC translocated to the membrane in unstimulated cells and PdBu further stimulated translocation of PKC to the membrane. Amounts of all four isoforms in cytoplasm and the membrane tended to be less in bIFN- treated cells. However, a clear translocation of PKC isoforms in the absence of PdBu in control cells suggests that translocation of PKC is not likely to be a major site for bIFN- induced suppression of PGF secretion. Cytosolic phospholipase A₂ (cPLA₂) was present in the cytoplasm and was translocated to the membrane in response to PdBU. Treatment with bIFN- blocked PdBU induced translocation of cPLA₂ and decreased cPLA₂ protein. The second objective determined if bIFN- suppression of PdBu stimulated PGF secretion is dependent upon protein synthesis. After 24 h starvation, BEND cells were pretreated with PdBu (100 ng/ml) for 3h and then with PdBu alone (100 ng/ml), PdBu + bIFN- (50 ng/ml), PdBu + Cycloheximide (Cx; 1 g/ml) and PdBu + Cx + bIFN- for an additional 5h. Treatments with Cx blocked PdBu-stimulated secretion of PGF ( bIFN- ) by BEND cells. Thus PdBu induction of PGF secretion is dependent upon protein synthesis and bIFN- appears to inhibit PGF secretion through regulation of Cox-2 and cPLA₂ protein expression.

Keywords: PKC, bIFN- , PGF and Uterus

This abstract is being presented at: 8:00 AM in session:
Uterus/Oviduct I