Stimulation of cultured amnion cell prostaglandin endoperoxide H synthase activity by glucocorticoids and phorbol ester.

OBJECTIVE: The effects of a phorbol ester activator of protein kinase C and the glucocorticoids cortisol and dexamethasone on the enzyme activity of prostaglandin H synthase (cyclooxygenase) in confluent cultures of human amnion epithelial cells were tested. STUDY DESIGN: Amnion epithelial cells from spontaneously delivered placentas at term were isolated and grown in culture until confluent. The activity of the enzyme prostaglandin H synthase was determined in these cells with a well-characterized enzyme assay monitoring the conversion of arachidonic acid to prostaglandin E2. The Michaelis-Menten constant and maximum velocity were determined from the substrate velocity data by means of Lineweaver-Burk plots. RESULTS: Amnion cells lost most of their prostaglandin H synthase activity within 2 days of culturing. This activity could be restored when cells were treated with 12-O-tetradecanoyl phorbol-13-acetate, a phorbol ester activator of protein kinase C, or with the glucocorticoids cortisol and dexamethasone. Epidermal growth factor also increased the specific activity, whereas 17 beta-estradiol had no effect on the specific activity of the enzyme. There was a direct correlation between the specific activity of prostaglandin H synthase and the output of prostaglandin E2 by cells treated with these agonists. CONCLUSION: Our data support the view that increases in prostaglandin H synthase specific activity in intrauterine tissues can be caused by stimulation by specific agonists, and this in turn is responsible for enhanced prostaglandin output by these tissues.

Prostaglandin endoperoxide synthase kinetics in human amnion before and after labor at term and following preterm labor.

To determine whether the kinetics of prostaglandin endoperoxide synthase (PGHS, commonly known as cyclooxygenase) in human amnion change with labor onset or between preterm and term labor, a specific enzyme assay was developed and characterized. The assay was linear for time (0-8 min) and protein concentration (5-30 micrograms/250 microliters incubation volume). The optimum pH was 8.0-8.5, and the enzyme reaction reached saturation at 10-20 microM arachidonic acid. Flufenamic acid was more efficacious than ibuprofen in the presence of 1 mM tryptophan in inhibiting enzyme activity. The Km and Vmax of PGHS were determined in 10 amnions obtained at elective caesarean section before labor onset (CS) at 39.3 +/- 0.8 wk gestational age (mean +/- SD, range = 38.5-41 wk) and 9 amnions obtained following spontaneous labor and vaginal delivery (SL) at 39.6 +/- 0.8 wk (range = 38.5-41 wk). The Km values were 1.4 +/- 1.2 mumol/l (CS) and 2.2 +/- 1.5 mumol/l (SL) (not different). However, the Vmax increased significantly (p < 0.05) from 11 +/- 8 (CS) to 19 +/- 4 (SL) pg PGE2/micrograms protein/min. In eight preterm amnions obtained following spontaneous labor and delivery at 32.9 +/- 2.1 wk (range = 29-36 wk), the Km and Vmax were 2.0 +/- 1.2 mumol/l and 17 +/- 9 pg PGE2/micrograms protein/min, respectively. Neither of these values was different from those of CS or SL amnions. None of the preterm pregnancies displayed histological evidence of infection. These results suggest that an increase in the mean amnion PGHS maximum velocity occurs in association with the onset of labor at term. The mean Vmax of PGHS in amnions obtained from idiopathic preterm spontaneous deliveries is between the CS and SL term values, reflecting, perhaps, multiple etiologies for preterm delivery.

Regulation of prostaglandin synthesis in the human amnion.

An increase in prostaglandin synthesis by intrauterine tissues may be responsible for labour initiation and/or maintenance in humans. In all studies to date, the amnion is the intrauterine tissue whose prostaglandin output consistently increases with the onset of labour. This may be due, in part, to acute activation of the phospholipases A2 and C and to an increase in the specific activity of prostaglandin H synthase (PGHS). A number of factors exist in amniotic fluid, the fetal membranes, the decidua and the placenta that can increase PGHS specific activity. Some of these factors may increase PGHS enzyme activity by gene expression and protein synthesis. Preliminary evidence is presented that suggests the hypothesis that PGHS specific activity increases before the onset of labour rather than as a consequence of labour initiation, and that idiopathic preterm labour may frequently be associated with increased PGHS activity. Hence, activation of PGHS gene expression and/or protein synthesis may be causal for term and preterm labour.

Arachidonic acid incorporation into lipids of term human amnion.

There were no differences in the rate or amount of (1-14C)-labeled arachidonic acid incorporated into triacylglycerides, diacylglycerides, or any phospholipid species of freshly dispersed term human amnion cells obtained before or after labor. Both phosphatidylcholine and phosphatidylethanolamine incorporated 14C-arachidonic acid in proportion to their molar percent of total amnion phospholipids, but phosphatidylinositol incorporated three times as much 14C-arachidonic acid, suggesting either a rapid turnover in this specific phospholipid pool or a greater specificity for the transfer of arachidonoyl-coenzyme A to lysophosphatidylinositol. No or little competition of 14C-arachidonic acid incorporation into triacylglycerides or phospholipids occurred with palmitic acid, linoleic acid, or gamma-linolenic acid. However, dihomo-gamma-linolenic acid, eicosapentaenoic acid, and unlabeled arachidonic acid were effective inhibitors. We conclude that the term amnion has high acyl transferase activity, that no change in the basal activity of this enzyme occurs with the onset of labor, and that a specific acyl transferase exists for 20-carbon polyunsaturated fatty acids.

Identification of calmodulin-like activity in term human amnion: effect of calmodulin inhibitors on prostaglandin biosynthesis.

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further investigation of calmodulin effects upon specific enzymes in the PGE2 synthetic pathway will be necessary to elucidate a role for calmodulin in PGE2 production.