Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E2 is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E2 by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E2 than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E2; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E2 biosynthesis in this tissue.     

Biosynthesis of prostaglandin E 2 in human skin: subcellular localization and inhibition by unsaturated fatty acids and anti-inflammatory drugs

The biosynthesis of prostaglandin E(2) (PGE(2)) from [1-(14)C]arachidonic acid has been demonstrated in homogenates and subcellular fractions of human epidermis. This biosynthetic capacity is localized in the microsomal fraction, indicating the presence of an active prostaglandin synthetase system associated with membranes of the skin. The incorporation of (14)C from [1-(14)C]arachidonic acid into PGE(2) by the microsomal fraction was enhanced by EDTA. This apparent increase in (14)C incorporation into PGE(2) in the presence of EDTA could be due at least in part to its chelating properties of removing the divalent cations in the homogenate that enhance the selective formation of PGF(2alpha) and the suppression of the activity of epidermal phospholipase A, which causes the release of nonradioactive fatty acid precursors from endogenous phospholipids. This study has also demonstrated that the formation of PGE(2) from arachidonic acid by the microsomal fraction from human skin could be inhibited by polyunsaturated fatty acids, suggesting a possible regulatory role of fatty acids released from endogenous phospholipids on prostaglandin synthesis in this tissue. The inhibitory effects of some anti-inflammatory drugs on skin microsomal prostaglandin synthetase were also demonstrated in these studies. Results from these studies indicate that the skin is therefore a useful tissue for the study of mechanisms of prostaglandin biosynthesis and the mode of action of various anti-inflammatory drugs.     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.     

Biphasic effects of nonsteroidal anti-inflammatory drugs on prostaglandin production by cultured rat calvariae

We have studied the effects on bone of three structurally dissimilar non-steroidal anti-inflammatory drugs which inhibit prostaglandin cyclo-oxygenase activity (PGH synthase); indomethacin, flurbiprofen, and piroxicam. We used cultures of half calvaria from neonatal or fetal rats to measure effects on PGE2 production, measured by radioimmunoassay. In four day neonatal rat calvaria, indomethacin inhibited PGE2 release into the medium by 80% at 10(-8) M, while flurbiprofen and piroxicam produced similar inhibition at 10(-6) M. However, at 10(-10) M, treatment with all three compounds resulted in an increase in medium PGE2 concentration of 60 to 120%. To assess the mechanism of this effect, bones were labeled with [3H]-arachidonic acid, washed and cultured in the presence or absence of piroxicam. At 10(-6) M, piroxicam inhibited production of cyclo-oxygenase products and arachidonic acid release. However, at 10(-10) M, there was a substantial increase in labeled products, particularly PGE2, despite a further decrease in arachidonic acid release. In 21 day fetal rat cultures, flurbiprofen was found to increase PGE2 release both in control cultures and cultures which had been incubated with cortisol (10(-8) M) to reduce endogenous arachidonic acid release and supplied with exogenous arachidonic acid (10(-5) M) to provide a substrate. These results indicate that three potent inhibitors of PGH synthase can, paradoxically, increase prostaglandin production at low concentrations. The effect does not appear to be due to increased arachidonic acid release, and could be due to increased PGH synthase activity.     

Transforming growth factor-beta inhibits prostaglandin production in amnion and A431 cells

We studied the effect of transforming growth factor-beta (TGF-beta) on prostaglandin E2 (PGE2) production and mitogenesis in human amnion cells and compared the response in amnion cells with that in A431 cells. Both amnion cells and A431 cells respond to epidermal growth factor (EGF) with increased production of PGE2 whereas EGF promotes mitogenesis in amnion cells but not in A431 cells. In amnion cells, TGF-beta was not mitogenic, and did not alter the mitogenic response of cells to EGF. Treatment of amnion cells with TGF-beta did, however, cause a decrease in PGE2 production relative to untreated cells, although EGF stimulated PGE2 production was not attenuated. In A431 cells, TGF-beta acted to decrease PGE2 production relative to untreated cells and to attenuate the stimulation of PGE2 production effected by EGF. The inhibitory action of TGF-beta on PG production in amnion and A431 cells is contrary to the stimulation of PG production in mouse calvaria reported by others and is suggestive that the effect of TGF-beta on prostaglandin production, like its effect on growth, varies between different cell types. Inhibition of PG production by treatment of amnion or A431 cells with mefenamic acid did not alter thymidine incorporation into DNA in response to EGF; similarly, the addition of PGE2 or PGF2 alpha to culture media of amnion or A431 cells had no effect on mitogenesis (in the absence or presence of EGF). Based on these findings, we conclude that PG production and EGF action on proliferation (stimulation in amnion cells; inhibition in A431 cells) are dissociated.     

Amnion cell prostaglandin E2 stimulatory activity in chorion laeve-conditioned medium

In this study we demonstrate the presence of a stimulant(s) to amnion cell prostaglandin (PG) E2 production in chorion-conditioned medium (CCM). The CCM induced a dose-dependent increase in amnion cell PGE2 production. This stimulatory activity was eliminated by heat and protease treatment. Maximal stimulation of amnion PGE2 by CCM did not occur until after 2 h of incubation, and treatment with cycloheximide (1 microgram/ml) effectively eliminated the ability of the amnion cells to respond to CCM. Additionally, CCM and arachidonic acid (2-40 microM) were synergistic in their stimulatory actions on amnion PGE2 production. CCM-treated amnion cells recover more quickly from acetylsalicylic acid pretreatment as compared to control. It is concluded that CCM contains a heat-labile protein which stimulates amnion cell PGE2 production by induction of prostaglandin endoperoxide synthase activity.     

Characterization of prostaglandin formation by human amnion cells in monolayer culture

In the present investigation, we found that among the prostanoids that human amnion cells, which are maintained in monolayer culture, secrete into the culture medium, prostaglandin E₂ is by far the predominant one. In the presence of inhibitors of prostaglandin synthase, the production of prostaglandin E₂ by these cells is abolished. Amnion cells maintained in the presence of fetal calf serum produce greater quantities of prostaglandin E₂ than do cells maintained in serumless medium. In the amnion cells, there is little or no metabolism of prostaglandin E₂; this also is true of amnion tissue. The unique characteristics of prostaglandin biosynthesis and metabolism by human amnion cells in monolayer culture are identical with those of human amnion tissue. Hence, we suggest that amnion cells in culture constitute an excellent model for investigations of the regulation of prostaglandin E₂ biosynthesis in this tissue.     

Phorbol ester-induced stimulation of prostaglandin biosynthesis in human amnion cells

Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.     

Dexamethasone stimulates arachidonic acid conversion to prostaglandin E2 in human amnion cells

The purpose of this investigation was to study the mechanism of stimulation of PGE2 output from human amnion epithelial cells by the synthetic glucocorticoid dexamethasone. Cells incubated in serum-free pseudo-amniotic fluid produced very low levels of PGE2, even when arachidonic acid (1 microM) was present. Pretreatment of cells with dexamethasone (50 nM) for 21 h increased the PGE2 output 6- to 7-fold in 2-h incubations only in the presence of arachidonic acid. The RNA synthesis inhibitor, actinomycin D (1 microgram/ml), and the protein synthesis inhibitor, cycloheximide (40 micrograms/ml), each blocked dexamethasone-stimulated arachidonic acid conversion to PGE2. The time course of these events suggests that dexamethasone first initiates RNA synthesis. Acetylsalicylic acid, a specific and irreversible blocker of prostaglandin endoperoxide H synthase (cyclooxygenase), was used to determine whether dexamethasone could stimulate new enzyme synthesis. Cells treated first with acetylsalicylic acid (30 min) then dexamethasone (22 h) produced as much PGE2 in response to 1 microM arachidonate as did cells exposed to dexamethasone only. Exposing cells to acetylsalicylic acid after dexamethasone completely eliminated PGE2 output. These data suggest that dexamethasone stimulates the synthesis of prostaglandin endoperoxide H synthase.     

Interleukin-4 differentially regulates prostaglandin production in amnion-derived WISH cells stimulated with pro-inflammatory cytokines and epidermal growth factor.

Cytokines and growth factors have been proposed to act as in vivo modulators of amnion prostaglandin production at parturition. To characterize the effects of the 'anti-inflammatory' cytokine interleukin (IL)-4 on amnion prostaglandin production, amnion epithelium-derived WISH cells were treated with IL-4 in the presence/absence of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or epidermal growth factor (EGF). IL-4 (0.08-10 ng/ml) potently inhibited cytokine-stimulated PGE2 production over 16 h (maximal inhibition approximately 66% at 2.0 ng/ml IL-4). Delaying addition of IL-4 (1 ng/ml) by up to 8 h after IL-1beta addition only slightly attenuated its inhibitory effects, from approximately 65% to approximately 50%. EGF-stimulated PGE2 production was either not inhibited or slightly stimulated by IL-4. Immunoblotting studies revealed that IL-4 (10 ng/ml) significantly suppressed prostaglandin-H synthase-2 (PGHS-2) levels in cells stimulated with IL-1beta and TNF-alpha over 16 h, but had no consistent effects on cytosolic phospholipase A2 (cPLA2) levels under any condition. In the presence of arachidonic acid (10 microM), IL-4 again inhibited cytokine-stimulated, but not EGF-stimulated, PGE2 production. The presence of IL-4 also failed to alter the amount of arachidonic acid released in response to EGF. These findings suggest a role and potential therapeutic application for IL-4 in inhibiting amnion PGHS-2 expression and hence prostaglandin production in infection-driven preterm labour, but not labour in the absence of inflammatory initiators.     

Evidence for a bidirectional prostaglandin endoperoxide shunt between platelets and the bovine coronary artery

While platelets have been shown to be capable of supplying prostaglandin (PG) H2 to endothelial cells in culture for PGI2 synthesis, endothelial cells have been shown unable to supply PGH2 to platelets for thromboxane (TX) A2 synthesis. We incubated rings of the bovine coronary artery (BCAR) with human platelets treated with aspirin (to inhibit cyclooxygenase) or CGS 13080 (to inhibit TXA2 synthase) in the presence of 20 microM arachidonic acid. BCAR, with damaged endothelium, produced significantly less PGI2 than that with intact endothelium. However, co-incubation with CGS 13080-treated platelets resulted in an increase in PGI2 independent of endothelium, demonstrating a shunt of PGH2 from platelets to BCAR. Co-incubation of BCAR with aspirin-treated platelets resulted in a net increase in TXA2 demonstrating a shunt of PGH2 from BCAR to platelets. Employing [14C]PGH2 as substrate, BCAR with and without intact endothelium produced similar amounts of 6-keto-[14C]PGF1 alpha. Likewise, homogenates (50 micrograms protein) of intimal and subintimal regions of BCAR and BCAR converted similar amounts of PGH2 to 6-keto-PGF1 alpha. These data suggest that vascular production of PGH2 is more dependent on an intact endothelium than is the conversion of PGH2 to PGI2. These data also suggest a potential for a bidirectional exchange of PGH2 between platelets and vascular wall during platelet-vascular wall interactions.     

Metabolism of arachidonic acid and prostanoids in human endometrial stromal cells in monolayer culture

In the present investigation, we compared the metabolism of arachidonic acid in human endometrial stromal cells maintained in monolayer culture with that in human decidual tissues. By gas-chromatographic analysis, the distribution of arachidonic acid in glycerophospholipids and in the neutral lipids of decidual tissues and stromal cells in culture was similar. After the addition of [14C]arachidonic acid to the culture medium, steady-state conditions with respect to radioactive labeling of the lipids of the cells were attained after 24 h, except for phosphatidylethanolamine and neutral lipids. The percentage distribution of [14C]arachidonic acid in the lipids of the cells in culture was as follows: phosphatidylcholine, 41%; phosphatidylserine, 5%; phosphatidylinositol, 19%; phosphatidylethanolamine, 22%; neutral lipids, 11%. This distribution of arachidonic acid among the lipids is similar to that in decidual tissue, except for that in phosphatidylethanolamine. The amount of radioactivity in phosphatidylethanolamine continued to increase up to 72 h whereas that in neutral lipids declined after a maximum amount was present at 4 h. In the cells in monolayer culture, [14C]prostaglandin E2 and [14C]prostaglandin F2 alpha were produced from [14C]arachidonic acid, as is true in superfused decidual tissue. The similarities in arachidonic acid metabolism in these cells to that in decidual tissue are supportive of the proposition that endometrial stromal cells in monolayer culture are an appropriate model for the study of the regulation of arachidonic acid release and prostaglandin formation by endometrium and decidua vera.     

Expression of prostaglandin G/H synthase (cyclooxygenase) during murine fetal thymic development.

Fetal thymic lobes in organ culture have been shown to have the capacity to metabolize [14C]arachidonic acid (AA) to prostaglandins (PGs), including 6-ketoPGF1 alpha, PGF2 alpha, PGE2, and PGA2. Inhibition of AA metabolism results in inhibition of growth and Thy 1 expression during thymic organ culture. We report herein that freshly-isolated fetal thymic lobes also have the capacity to metabolize [14C]AA to PGs and HETEs at Days 14 and 16 of prenatal murine development. RNA encoding phospholipase A2, which liberates arachidonic acid from membrane phospholipids, and cyclooxygenase (prostaglandin G/H synthase), the first enzyme involved in the conversion of AA to PGs, are expressed during thymic development. We have localized the cyclooxygenase protein to stromal cells in the fetal and adult thymus. Exogenous AA or an analogue of PGI2 (iloprost) stimulated growth of fetal thymocytes in organ culture. These findings, together with our studies of the morphology of thymic lobes cultured with inhibitors of arachidonate metabolism, support the hypothesis that PGs are required for thymocyte proliferation during thymic development.     

Regulation of prostaglandin H synthase activity in dog urothelial cells.

TPA regulation of prostaglandin H synthase activity in primary and subcultured dog urothelial cells was investigated. Previous studies have demonstrated an early (0-2 hr) increase in PGE2 synthesis mediated by TPA which is dependent upon release of endogenous arachidonic acid by a phospholipase-mediated pathway. In this study, prostaglandin H synthase activity was assessed directly with microsomes and indirectly after addition of exogenous arachidonic acid at a maximum effective concentration (100 microM) to media. PGE2 synthesis, measured by radioimmunoassay, served as an index of prostaglandin H synthase activity. After a 24-hr incubation with 0.1 microM TPA or 1.0 microM A23187, arachidonic acid elicited significantly more PGE2 synthesis in agonist-treated cells than it did in control cells in primary culture. Microsomes from 24-hr TPA-treated cells exhibited significantly more prostaglandin H synthase activity than did those from control cells. In addition, the PGE2 content of overnight media was approximately 10-fold greater in TPA-treated cells than in control cells. The late (24 hr) response was more sensitive to lower concentrations of TPA than was the earlier (0-2 hr) response. TPA at 0.1 microM was a maximum effective dose for both responses. The 24-hr response was blocked by cycloheximide and staurosporine, inhibitors of protein synthesis and protein kinase C, respectively. Pretreatment of cells with aspirin, an irreversible inhibitor of prostaglandin H synthase, prior to addition of TPA did not prevent the late TPA-mediated increase in PGE2 synthesis. Subcultured cells exhibited both an early and a late TPA response. Only the early response was inhibited by aspirin pretreatment. Results suggest that the late response with TPA is caused by de novo synthesis of prostaglandin H synthase. Thus, primary and subcultured dog urothelial cells possess two distinct mechanisms for regulating signal transduction by arachidonic acid metabolism. This study provides a basis for assessing these mechanisms of signal transduction in urothelial cell lines and transformed cells.     

The regulation of prostaglandin biosynthesis in cells derived from human gestational tissues: effects of fetal calf serum.

We have evaluated the production of prostaglandin E2 (PGE2) and its regulation in amnion, chorion, and decidual cells in the presence and absence of fetal calf serum (FCS), and in the absence of FCS but with supplementation with substrate arachidonic acid (AA). Basal rates of PGE2 biosynthesis in amnion, chorion and decidual cell cultures were significantly reduced in the absence of FCS. The magnitudes of the responses to various stimulatory agents were different between cells from different tissues and the different culture media. We conclude that these different experimental conditions must be taken into account when interpreting the results of such in vitro experiments.     

Activity of prostaglandin biosynthetic pathways in rat pancreatic islets

Isolated pancreatic islets of the rat were either prelabeled with [3H]arachidonic acid, or were incubated over the short term with the concomitant addition of radiolabeled arachidonic acid and a stimulatory concentration of glucose (17mM) for prostaglandin (PG) analysis. In prelabeled islets, radiolabel in 6-keto-PGF1 alpha, PGE2, and 15-keto-13,14-dihydro-PGF2 alpha increased in response to a 5 min glucose (17mM) challenge. In islets not prelabeled with arachidonic acid, label incorporation in 6-keto-PGF1 alpha increased, whereas label in PGE2 decreased during a 5 min glucose stimulation; after 30-45 min of glucose stimulation labeled PGE levels increased compared to control (2.8mM glucose) levels. Enhanced labelling of PGF2 alpha was not detected in glucose-stimulated islets prelabeled or not. Isotope dilution with endogenous arachidonic acid probably occurs early in the stimulus response in islets not prelabeled. D-Galactose (17mM) or 2-deoxyglucose (17mM) did not alter PG production. Indomethacin inhibited islet PG turnover and potentiated glucose-stimulated insulin release. Islets also converted the endoperoxide [3H]PGH2 to 6-keto-PGF1 alpha, PGF2 alpha, PGE2 and PGD2, in a time-dependent manner and in proportions similar to arachidonic acid-derived PGs. In dispersed islet cells, the calcium ionophore ionomycin, but not glucose, enhanced the production of labeled PGs from arachidonic acid. Insulin release paralleled PG production in dispersed cells, however, indomethacin did not inhibit ionomycin-stimulated insulin release, suggesting that PG synthesis was not required for secretion. In confirmation of islet PGI2 turnover indicated by 6-keto-PGF1 alpha production, islet cell PGI2-like products inhibited platelet aggregation induced by ADP. These results suggest that biosynthesis of specific PGs early in the glucose secretion response may play a modulatory role in islet hormone secretion, and that different pools of cellular arachidonic acid may contribute to PG biosynthesis in the microenvironment of the islet.     

Effects of fetal urinary corticosteroids, catecholamines and kallikrein on PGE2 synthesis in monolayer cultures of human amnion and chorion cells

We have studied the effects of several compounds isolated from fetal urine on the production of PGE2 by amnion and chorion cells which were maintained in primary monolayer culture. We conclude that desoxycorticosterone and vanillylmandelic acid stimulate PGE2 synthesis by amnion, but adrenaline and kallikrein increase the biosynthesis of PGE2 by chorion. These data suggest that human fetal urine could play a major role in events of parturition.     

Paradoxical stimulation of both lipocortin and prostaglandin production in human amnion cells by dexamethasone

Glucocorticoids inhibit prostaglandin biosynthesis by inducing the formation of lipocortins. In human amnion cells dexamethasone elicited a concentration-dependent increase in prostaglandin production and raised intracellular lipocortin 1 concentrations. Dexamethasone could also potentiate the epidermal growth factor (EGF)-induced stimulation of prostaglandin production. EGF alone or in combination with dexamethasone increased lipocortin 1 formation in amnion cells. Human amnion cells may provide a unique insight into interactions between glucocorticoids, lipocortin and eicosanoid biosynthesis.