Synthesis and release of eicosanoids by amnion in response to a fetal or maternal signal may be involved the onset of parturition in humans but the intracellular signal transduction mechanisms that lead to eicosanoid synthesis (the response) in amnion are ill-defined. Delineation of a transduction mechanism requires identification of substrates that are covalently modified, the specific modifications made and demonstration of a cause and effect relationship. We have evidence that metabolism of arachidonic acid switches from lipoxygenase to cyclo-oxygenase pathways at parturition and that activation of protein kinase C (PKC) may be involved in the transduction mechanism that stimulates PGE 2 synthesis in amnion. PKC may phosphorylate key regulatory elements which either control arachidonic acid mobilization or its subsequent metabolism to eicosanoid. the elements involved in mobilization include phospholipase enzyme activity and the phospholipase inhibitors, the lipcortins, which have been shown to be activated or de-inhibited, respectively, by phosphorylation. The elements involved in arachidonic acid metabolism, about which nothing is known regarding their activity in response to phosphorylation, include the PGH 2 synthase/PGE 2 isomerase and lipoxygenase enzymes.
We aim to: 1. Precisely identify the lipoxygenase metabolites produced by amnion, examine if production changes at parturition and if alterations in concentrations or activies of lipoxygenase and PGH2 synthase/ PGE2 isomerase enzymes occur. 2. Identify the phosphoproteins involved in the transduction mechanism, specifically which isoform(s) of lipocortin and phospholipase are present in amnion, their specific PKC phosphorylation sites and if phosphorylation increases phospholipase activity and hence PG production. Examine if phosphorylation of these isoforms occurs at parturition and fits the criteria necessary to be part of a signal transduction mechanism. 3. Establish the role of PKC in calcium ionophore-stimulated amnion PGE2 synthesis and the identity of the phosphoproteins that may be involved. 4. Determine whether putative stimulants of amnion PGE2 synthesis e.g. EGF, TGFa, IL-1 and PAF act via the PKC-mediated pathway outlined above. Once the transduction mechanism that controls arachidonic acid metabolism is precisely defined then the real endogenous in vivo signal that initiates it can be defined. The opportunities for prevention of preterm labor at the biochemical level then become real.
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