Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.     

Insulin and leptin do not affect fatty acid uptake and metabolism in human placental choriocarcinoma (BeWo) cells

Placental transport of long chain polyunsaturated fatty acids is important for fetal growth and development. In order to examine the effects of leptin and insulin on fatty acid uptake by the placenta, placental choriocarcinoma (BeWo) cells were used. BeWo cells were incubated for 5h at 37 degrees C in the absence or presence of different concentrations of insulin (0.6, 60, and 100 ng) or leptin (10 ng) with 200 microM of various radiolabeled fatty acids (docosahexaenoic acid, arachidonic acid, eicosapentaenoic acid, and oleic acid, mixed with 1:1 bovine serum albumin (fat free). After incubation, the uptake and distribution of these fatty acids into different cellular lipid fractions were determined. The uptakes of oleic, eicosapentaenoic, arachidonic, and docosahexaenoic acids were 15.36+/-4.1, 19.95+/-3.6, 28.56+/-8.1, and 62.25+/-9.5 nmol/mg of protein, respectively, in BeWo cells. Incubation of these cells with insulin (0.6 or 60 ng/ml) or leptin (10 ng/ml) did not significantly alter uptake of any of these fatty acids (P>0.5). Insulin or leptin also did not affect beta oxidation of fatty acids in these cells. In contrast, leptin (10 ng/ml) and insulin (0.60 ng/ml)) stimulated the uptake of oleic acid (7.4+/-2.3 nmol/mg protein) in human adipose cells, SGBS cells by 1.28- and 2.48-fold (P<0.05), respectively. The distribution of fatty acids in different cellular lipid fractions was also not affected by these hormones. Our data indicate that unlike adipose tissue, fatty acid uptake and metabolism in placental trophoblasts is not regulated by insulin or leptin.     

Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells

We have investigated the extent to which modifications in the essential fatty acid content of mammalian cells can affect prostaglandin production. Swiss mouse 3T3 cells stimulated with the calcium ionophore A23187 produced 1.7 to 7 times more prostaglandin E(2) (PGE(2)) when the cultures were supplemented with linoleic acid. Increases in PGE(2) production as a result of linoleic acid supplementation occurred under all culture conditions except during the first 24 hr after attachment, when prostaglandin production was very high. Arachidonic acid supplementation produced a similar enhancement in the capacity of the cells to produce PGE(2), but no appreciable increase occurred when the cultures were supplemented with oleic acid. The phospholipids of the cells exposed to the linoleate-enriched medium contained 4 times more arachidonic acid and twice as much linoleic acid as compared with the corresponding controls. The choline phosphoglycerides were most highly enriched in arachidonic acid, but 2- to 3-fold increases also occurred in the inositol and ethanolamine phosphoglycerides. When cultures initially enriched with linoleic acid were transferred to an unsupplemented medium, the fatty acid composition as well as the capacity of the cells to produce PGE(2) reverted almost to control values. The amount of exogenous arachidonic acid converted to PGE(2) as measured by radioimmunoassay also was greater when the cells were enriched with linoleic acid. Studies with radioactive arachidonic acid indicated that the distribution of prostaglandin metabolites was not affected appreciably by linoleic acid enrichment. These findings suggest that at least two factors contribute to the increased capacity of the cultures supplemented with linoleate to produce PGE(2). One is enrichment of the phospholipid substrate pools with arachidonic acid. The other is an increased ability of the cells to synthesize PGE(2) from unesterified arachidonic acid, perhaps because the prostaglandin-forming enzymes are more active.-Denning, G. M., P. H. Figard, and A. A. Spector. Effect of fatty acid modification on prostaglandin production by cultured 3T3 cells.     

Effect of diarachidonin on prostaglandin E2 synthesis in rabbit kidney medulla slices.

The effect of diarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. The addition of diarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. At three concentrations (10, 50 and 100 microM), increases in prostaglandin E2 formation induced by exogenous diarachidonin were 2-fold greater than those induced by exogenous arachidonic acid. Diacylglycerol or phosphatidic acid from egg lecithin had little or no effect on prostaglandin E2 production. Moreover, EGTA failed to inhibit diarachidonin-stimulated prostaglandin E2 formation, indicating that the stimulatory effect of diarachidonin is not mediated through the activation of endogenous phospholipase A2 (including phosphatidic acid-specific phospholipase A2). These results are discussed in the light of our former hypothesis that arachidonic acid release from kidney medulla phospholipids might occur through the sequential action of a phospholipase C coupled to diacylglycerol and monoacylglycerol lipases [Fujimoto, Akamatsu, Hattori & Fujita (1984) Biochem. J. 218, 69-74].     

Okadaic acid and dinophysistoxin-1, non-TPA-type tumor promoters, stimulate prostaglandin E2 production in rat peritoneal macrophages

Okadaic acid and dinophysistoxin-1 isolated from a black sponge, Halichondria okadai are non-12-O-tetrade-canoylphorbol 13-acetate (non-TPA)-type tumor promoters of mouse skin. Okadaic acid at concentrations of 10-100 ng/ml stimulated prostaglandin E2 production in rat peritoneal macrophages. Dinophysistoxin-1 (35-methylokadaic acid) stimulated prostaglandin E2 production as strong as okadaic acid, but okadaic acid tetramethyl ether, an inactive compound as a tumor promoter, did not. Okadaic acid at 10 ng/ml (12.4 nM) stimulated prostaglandin E2 production as strongly as TPA at 10 ng/ml (16.2 nM) 20 h after incubation. Unlike TPA-type tumor promoters, okadaic acid required a lag phase before stimulation. The duration of this lag phase was dependent on the concentration of okadaic acid. Indomethacin inhibited okadaic acid-induced preostaglandin E2 production in a dose-dependent manner, and its inhibition was more strongly observed in okadaic acid-induced prostaglandin E2 production. Cycloheximide inhibited okadaic acid-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages and prostaglandin E2 production dose dependently, suggesting that protein synthesis is a prerequisite for the stimulation of arachidonic acid metabolism. These results support our idea that tumor promoters, at very low concentrations, are able to stimulate arachidonic acid metabolism in rat peritoneal macrophages.     

Stimulation of prostaglandin E2 production by phorbol esters and epidermal growth factor in porcine thyroid cells

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.     

Microtubule disruption enhances prostaglandin E2 production in osteoblastic cells

Prostaglandins have been implicated in the response of bone to mechanical stimuli. To explore the potential role of the cytoskeleton in the control of prostaglandin production, we examined the effect of cytoskeleton disrupting agents on arachidonic acid metabolism in rat calvaria osteoblastic cells. We found that microtubule disrupting agents increase prostaglandin E production 4-5-fold. Stimulation was first detectable at 4 h and rose sharply between 4 and 8 h. 2 h exposure to 1 microM colchicine was sufficient to produce the maximum effect. Cytochalasin B at concentrations which caused marked shape changes had no effect on prostaglandin E production or on its stimulation by colchicine. Taxol, a stabilizer of microtubules, reduced the colchicine effect. The increase in prostaglandin E production was associated with enhanced conversion of arachidonic acid to prostaglandin E2 rather than enhanced release of arachidonic acid from phospholipids. This increase in enzymatic activity was not abolished by cycloheximide treatment at concentrations which inhibited 90% of protein synthesis in the cells.     

Effect of essential polyunsaturated fatty acid modifications on prostaglandins production by MDCK canine kidney cells

Supplementation of growing MDCK canine kidney tubular epithelial cultures with linoleic acid produced a 3.6- to 4.9-fold increase in bradykinin-stimulated PGE₂ release as measured by radioimmunoassay. Under these conditions the cell phospholipids contained 3.9-times more linoleic acid and 5.6-times more arachidonic acid, with the inositol, ethanolamine and choline phosphoglycerie fractions becoming enriched in arachidonic acid. By contrast, supplementation with arachidonic acid did not enhance bradykinin-stimulated PGE₂ release even though the arachidonic acid content of the cell phospholipids was increased 8.8-fold. The distribution of radioactive prostaglandin products was unchanged by these fatty acid enrichments, with PGE₂ accounting for 55 to 68% of the total output from [1-¹⁴C]arachidonic acid. Linoleic acid supplementation also produced a 2.5-fold increase in PGE₂ formation stimulated by extracellular arachidonic acid, whereas supplementation during culture with arachidonic acid caused a 55 to 80% inhibition. This difference cannot be accounted for by changes in the ability of the cells to incorporate extracellular arachidonic acid. it is suggested that at least some of the effects of linoleate supplementation on prostaglandin production are due to the resulting enrichment of the intracellular phospholipid substrate pools with arachidonic acid. In addition, it appears that prolonged exposure to arachidonic acid during culture has an overriding inhibitory effect on prostaglandin production even though the total cell lipids bocome highly enriched in arachidonate.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

Prostaglandin generation in rabbit kidney. Hormone-activated selective lipolysis coupled to prostaglandin biosynthesis.

The endogenous release of prostaglandins and free fatty acids from the isolated perfused rabbit kidney in the absence or presence of stimulation by bradykinin or angiotensin-II was investigated. Basal (nonstimulated) release of prostaglandin-precursor arachidonic acid was 15-20-fold higher than that of prostaglandin E2 indicating a low conversion of released arachidonate to prostaglandins. Addition of bovine serum albumin to the perfusion medium caused a substantial (50-250%) increase in the release of all fatty acids except myristic and arachidonic acids, and no significant change in prostaglandin E2 generation. In contrast, administration of bradykinin (0.5 microgram) or angiotensin-II (1 microgram) caused a 10-15-fold increase in prostaglandin E2 release, and with albumin present, also a 2-3-fold selective increase in arachidonic acid release. Thus, unlike what was observed under basal conditions, arachidonic acid released following hormone stimulation is efficiently converted to prostaglandin E2. We conclude that administration of bradykinin or angiotensin-II into the perfused kidney activates a lipase which selectively releases arachidonic acid, probably from a unique lipid entity. This lipase reaction is tightly coupled to a prostaglandin generating system so that the released arachidonate is first made available to the prostaglandin cyclooxygenase, resulting in its substantial conversion to prostaglandins.     

Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor

Linoleic acid, arachidonic acid, prostaglandin E1, and prostaglandin E2 stimulated the proliferation of mammary epithelial cells in serum-free primary cultures only in the presence of epidermal growth factor. Linoleate-stimulated growth was manifest later in culture when proliferation, initiated by epidermal growth factor only, reached a plateau while linoleate-supplemented epidermal growth factor cultures continued to proliferate. The cultures in the plateau phase of growth could be restimulated to grow by adding either linoleic acid or prostaglandin E2 to the media. While the linoleate response could be abolished by the cyclooxygenase inhibitor, indomethacin, prostaglandin E2-stimulated growth remained unaffected. Linoleic acid was metabolized to arachidonic acid and prostaglandin E2, both in the growing and resting cultures. Proliferating cells metabolized linoleate and prostaglandin E2 extensively so that neither the fatty acid nor prostaglandin E2 accumulated in large quantities in the proliferating cultures. The concentrations of prostaglandin E2 in growing cultures supplemented with linoleic acid were much higher than in cultures without it. These results suggest that the metabolism of linoleic acid leading to prostaglandin production, not its contribution to membrane polyunsaturation, is necessary for sustained growth of mammary epithelial cells in the presence of epidermal growth factor.     

Early changes in inositol lipids and their metabolites induced by platelet-derived growth factor in quiescent Swiss mouse 3T3 cells.

Inositol lipid turnover was studied in quiescent Swiss mouse 3T3 cells stimulated by platelet-derived growth factor (PDGF). Stimulation of the cells by PDGF for 10 min at 37 degrees C induced the following changes in lipids: in cells prelabelled with [32P]Pi, a 28% decrease in [32P]phosphatidylinositol 4,5-bisphosphate, a 41% decrease in [32P]phosphatidylinositol 4-phosphate and a 1.7-fold increase in the 32P-labelling of phosphatidic acid; in cells prelabelled with [3H8]arachidonic acid, a 17.9-fold increase in [3H]phosphatidic acid, a 20% decrease in [3H]phosphatidylinositol (PtdIns), an 8.6-fold increase in [3H]arachidonic acid released into the medium, a 57-fold increase in [3H]prostaglandin E2 in the medium, and a 5.3-fold increase in [3H]monoacylglycerol released into the medium (the last was identified as the 2-acyl derivative); in cells prelabelled with [2-3H]glycerol, a 1.7-fold increase in [3H]diacylglycerol, a 6.7-fold increase in [3H]phosphatidic acid, a 1.6-fold increase in [3H]lysophosphatidylcholine (lysoPtdCho), a 9% decrease in [3H]PtdIns, and a 1.6-fold increase in [3H]monoacylglycerol released into the medium. PDGF stimulated the formation of inositol tris-, bis- and mono-phosphates in the cells prelabelled with myo-[2-3H]inositol. These results indicate that, in Swiss 3T3 cells stimulated by PDGF, diacylglycerol produced by the hydrolysis of inositol lipids is partly degraded to 2-acylglycerol and partly converted into phosphatidic acid. The increase in lysoPtdCho indicates that a portion of arachidonic acid released from the stimulated cells is formed by the hydrolysis of PtdCho with a phospholipase A2. Different values of half-maximal doses of the partially purified PDGF used in this study were found for the various responses of quiescent Swiss 3T3 cells to PDGF. The values for half-maximal doses suggest that activation of a fraction of the cell-surface receptor for PDGF is sufficient for mitogenesis and for an increase in the cytoplasmic free Ca2+ concentration, and that the PGDF-stimulated lipid metabolism is probably proportional to the number of receptor sites activated by PDGF.     

Prostaglandin synthesis in human placenta and fetal membranes.

The conversion of exogenous arachidonic acid into prostaglandins was studied in human placenta and fetal membrane microsomes. Only one prostaglandin was formed, prostaglandin E2 (PGE2), in fetal membrane microsomes. In placental microsomes PGE2 was further transformed into 15 keto-PGE2. Cofactor requirements and some characteristics of the system were studied. 1 to 3% conversion of arachidonic acid into prostaglandins was observed in placental microsomes and 5 to 8% conversion in fetal membrane microsomes.     

Effect of prostaglandins and their analogues on hormone-stimulated glycogenolysis in primary cultures of rat hepatocytes

Hepatocytes were isolated by collagenase perfusion method from adult male rats, cultured and then prelabeled with [14C]glucose. The [14C]glycogen-labeled cells were used in experiments for effect of prostaglandins on hormone-stimulated glycogenolysis. Prostaglandin E1, prostaglandin E2 and 16,16-dimethylprostaglandin E2, but not prostaglandin D2 or prostaglandin F2 alpha, inhibited glycogenolysis stimulated by glucagon, epinephrine, isoproterenol (beta-adrenergic agonist) or epinephrine in the presence of propranolol (beta-antagonist) in primary cultured hepatocytes. The inhibitory effects on day 2 of cultures were approx. twice those on day 1. Dimethylprostaglandin E2 (10(-6)M) caused 60-70% inhibitions of the stimulations by these substances. In the case of the stimulation by glucagon, the inhibition further increased by 80-100% on day 3 of culture. Prostaglandin E1 and prostaglandin E2 caused less inhibition than dimethylprostaglandin E2 of all these stimulations. Dinorprostaglandin E1 (9 alpha,13-dihydroxy-7-ketodinorprost-11-enoic acid), which is a hepatocyte-metabolite of prostaglandin E1 and prostaglandin E2, and arachidonic acid did not have any inhibitory effects. These data indicate that the E series of prostaglandins may function as the regulation of hepatic glycogenolysis stimulated by epinephrine and glucagon, and that their rapid degradation system may contribute to the modulation of the action in liver.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Stimulation of prostaglandin E2 production by 12-O-tetradecanoylphorbol 13-acetate (TPA)-type and non-TPA-type tumor promoters in macrophages and its inhibition by cycloheximide

The effects of TPA (12-O-tetradecanoylphorbol 13-acetate)-type and non-TPA-type tumor promoters on prostaglandin E2 production by peritoneal macrophages of rats were examined. Among the TPA-type tumor promoters, aplysiatoxin was most potent in stimulating prostaglandin E2 production followed by dihydroteleocidin B, teleocidin, TPA and debromoaplysiatoxin. Prostaglandin E2 production by aplysiatoxin treatment was stimulated at doses up to 0.1 ng/ml. Palytoxin, a non-TPA-type tumor promoter, also stimulated both prostaglandin E2 production and the release of radioactivity from [3H]arachidonic acid-labeled macrophages. However, the dose required for the expression of these effects by palytoxin was up to 3 pg/ml. It was suggested that the tumor promoters are associated with the activity to stimulate arachidonic acid metabolism, irrespective of their type. Cycloheximide, a protein synthesis inhibitor, inhibited both prostaglandin E2 production and the release of radioactivity from prelabeled macrophages stimulated either by the TPA-type tumor promoters or by the non-TPA-type tumor promoter. It is possible that the tumor promoters may induce the synthesis of some proteins responsible for the stimulation of arachidonate metabolism.     

Elevated prostaglandin synthetase activity in methylcholanthrene-transformed mouse BALB/3T3

Cell lines transformed from 3T3 spontaneously, by radiation, or by treatment with chemical carcinogens, polyoma and SV40 virus produce up to 5 times more prostaglandins than their untransformed parent line. Several aspects of prostaglandin biosynthesis by MC5-5 and 3T3 were compared. When stimulated by serum, bradykinin, or thrombin, MC5-5 produced 2-to 5-fold more prostaglandins than 3T3. With the use of cells labeled with radioactive arachidonic acid in their cellular lipids, these higher levels were shown not to be due to increased availability of the prostaglandin precursor, arachidonic acid. Prostaglandin synthetase activity in microsomal fractions prepared from MC5-5 was 6 times higher than that of microsomes of untransformed cells. The increased prostaglandin levels produced by transformed cells therefore appear to be the result of elevated prostaglandin synthetase activity.     

Arachidonic acid is preferentially metabolized by cyclooxygenase-2 to prostacyclin and prostaglandin E2

The two cyclooxygenase isoforms, cyclooxygenase-1 and cyclooxygenase-2, both metabolize arachidonic acid to prostaglandin H2, which is subsequently processed by downstream enzymes to the various prostanoids. In the present study, we asked if the two isoforms differ in the profile of prostanoids that ultimately arise from their action on arachidonic acid. Resident peritoneal macrophages contained only cyclooxygenase-1 and synthesized (from either endogenous or exogenous arachidonic acid) a balance of four major prostanoids: prostacyclin, thromboxane A2, prostaglandin D2, and 12-hydroxyheptadecatrienoic acid. Prostaglandin E2 was a minor fifth product, although these cells efficiently converted exogenous prostaglandin H2 to prostaglandin E2. By contrast, induction of cyclooxygenase-2 with lipopol- ysaccharide resulted in the preferential production of prostacyclin and prostaglandin E2. This shift in product profile was accentuated if cyclooxygenase-1 was permanently inactivated with aspirin before cyclooxygenase-2 induction. The conversion of exogenous prostaglandin H2 to prostaglandin E2 was only modestly increased by lipopolysaccharide treatment. Thus, cyclooxygenase-2 induction leads to a shift in arachidonic acid metabolism from the production of several prostanoids with diverse effects as mediated by cyclooxygenase-1 to the preferential synthesis of two prostanoids, prostacyclin and prostaglandin E2, which evoke common effects at the cellular level.     

Alpha 1-adrenergic stimulation of arachidonic acid release and metabolism in a rat thyroid cell line. Mediation of cell replication by prostaglandin E2

The rat thyroid cell line, FRTL-5, expresses an alpha 1-adrenergic receptor when exposed to thyrotropin. We have found that occupation of this alpha 1-adrenergic receptor by norepinephrine stimulated the release of [3H]arachidonic acid from prelabeled cells. Arachidonic acid was metabolized primarily to prostaglandin E2 and to much smaller amounts of 11-hydroxy-5,8,11,13-eicosatetraenoic acid, 15-hydroxy-5,8,11,13-eicosatetraenoic acid, prostaglandin D2, and thromboxane B2. Synthesis of all these metabolites was inhibited by the cyclooxygenase inhibitor indomethacin. When FRTL-5 cells were starved of thyrotropin for 24 h, norepinephrine nearly doubled [3H]thymidine uptake into DNA. Cyclooxygenase inhibitors inhibited norepinephrine-stimulated thymidine uptake by 60-70%. Of several arachidonic acid metabolites tested, none was able to stimulate thymidine uptake directly in the presence of indomethacin. Prostaglandin E2, however, was able to restore [3H]thymidine uptake when added together with norepinephrine in the presence of indomethacin. Thus, occupation of an alpha 1-adrenergic receptor in a functional rat thyroid cell line leads to arachidonic acid release. Subsequent metabolism of the arachidonic acid by the cyclooxygenase pathway leads to synthesis of prostaglandin E2, which mediates a norepinephrine-stimulated activity related to cell replication.     

Epidermal growth factor stimulation of prostaglandin E2 biosynthesis in amnion cells. Induction of prostaglandin H2 synthase

Amnion is believed to be a tissue of signal importance, anatomically and functionally, in the maintenance of pregnancy and during the initiation of parturition. Epidermal growth factor (EGF)-like agents cause a striking increase in the secretion of prostaglandin E2 (PGE2) in human amnion cells but only if arachidonic acid is present in the culture medium. To investigate the regulation of arachidonic acid metabolism by EGF-like agents in amnion, we used mEGF and human amnion cells in primary monolayer culture as a model system. The amount of PGE2 secreted into the culture medium was quantified by radioimmunoassay and the rate of conversion of [14C]arachidonic acid to [14C]PGE2 (PGH2 synthase activity) in cell sonicates was determined under optimal in vitro conditions. Treatment of amnion cells with mEGF led to a marked increase in the rate of production of PGE2. The specific activity of PGH2 synthase (viz. the combined activities of prostaglandin endoperoxide (PGH2) synthase and PGH2-PGE isomerase) was increased by 2-5-fold in cells treated with mEGF. Treatment of amnion cells with mEGF for 4 h did not affect the specific activities of phospholipase A2 or phosphatidylinositol-specific phospholipase C. By immunoisolation of newly synthesized, [35S]methionine-labeled PGH2 synthase, we found that mEGF stimulated de novo synthesis of the enzyme. Thus, mEGF acts in human amnion cells in primary monolayer culture to increase the rate of PGE2 biosynthesis by a mechanism that involves induction of PGH2 synthase; the manifestation of EGF action on PGE2 biosynthesis is dependent on the presence of nonesterified arachidonic acid.