Effects of prostaglandin F2alpha and E2 on the production of progesterone by human granulosa cells in tissue culture.

Human granulosa cells with differing steroidogenic potentials were cultured in vitro. The effects of prostaglandin F2alpha (PGF2alpha) and PGE2 on the progesterone output and viability of these cells were investigated. Prostaglandin F2alpha either alone or in combination with LH and FSH inhibited the production of progesterone over a wide range of concentrations (1-8000 ng/ml). However, the inhibitory effect of PGF2alpha was 200 times less effective when the cells were exposed to LH and FSH for 6 days before the addition of the prostaglandin. By contrast PGE2, at concentrations from 1 to 500 ng/ml, markedly stimulated the production of progesterone by granulosa cells, and this was not prevented by the addition of PGF2alpha. The degree of inhibition by PGF2alpha or stimulation by PGE2 was related to the biosynthetic capacity of the cells. These studies suggest that PGF2alpha may act directly on the adenylate cyclase system of human granulosa cells by blocking its activation by LH, and they demonstrate that functional regression of the luteal cell can be induced independently of the blood vascular system.     

Effect of prostaglandins(PGs) on progesterone production by human cultured luteal cells and their ability of PGs production

The present study was designed to investigate whether or not prostaglandins(PGs) were produced by human luteal cells(HLC) and their effects on the luteal cells by monolayer culture. The following results were obtained. Cultured HLC secreted progesterone(P), prostaglandin F(PGF) and prostaglandin E(PGE) into a medium at concentrations of 276.6 +/- 38.6, 1.95 +/- 0.36, 2.44 +/- 0.45 ng/ml/1 X 10(5) cells/day (mean +/- SE), respectively. Cultured HLC was able to convert 14C-arachidonic acid to 14C-PGF2 alpha, 14C-PGE2. These two results indicated that HLC had the ability to produce PGF and PGE. Cultures were carried out in the presence of indomethacin (Ind), PGF2 alpha and PGE2 alone as well as in a combination. P production by HLC was reduced in the presence of Ind. P production in the presence of Ind+PGE2 was more than that in the presence of Ind alone. There was no significant difference in P production between the presence of Ind and Ind+PGF2 alpha. It was concluded that HLC had the ability to produce PGs and that PGE2 significantly stimulated P production in as low concentrations as HLC could produce physiologically while PGF2 alpha did not.     

Influence of 16-aryloxyprostaglandins on the production of progesterone by human granulosa cells in vitro.

16-Aryloxy analogues of prostaglandin F2alpha (PGF2alpha) are potent luteolysins in laboratory and farm animals. When their effect on progesterone production by luteinized human granulosa cells in tissue culture was investigated inhibition of both basal gonadotrophin-stimulated progesterone production was observed, so revealing characteristics expected of potential human luteolysins. The analogues were, however, unable to inhibit progesterone production stimulated by PGE2, suggesting that like PGF2alpha these compounds may act by specifically blocking LH-activated adenylate cyclase. The 16-aryloxyprostaglandins similarly inhibited progesterone production by porcine granulosa cells, so that the effects observed with the 16-aryloxyprostaglandins in vitro may be indicative of their potential in vivo.     

Stimulation of testosterone production in isolated rabbit thecal tissue by LH/FSH, dibutyryl cyclic AMP, PGE2alpha, and PGE2.

The capacities of isolated rabbit theca and granulosa cells to secrete testosterone were studied in vitro. Large Graafian follicles (1-1.5 mm in diameter) were dissected intact from the ovaries of adult estrous rabbits. Granulosa cells from 4 follicles (50,000 cells) and theca tissue (16 pieces per dish, equivalent to 4 follicles) were cultured separately for 6 days either as controls (without exogenous hormones) or with one of the following agents: 1 lU/ml LH/FSH (Pergonal), 10-3M dibutyryl cyclic AMP (Bu2cAMP), 1 mug/ml prostaglandin F2alpha (PGF2alpha), or 1 mug/ml prostaglandin E2 (PGE2). The media were collected every 2 days, and the testosterone (T) was measured by radioimmunoassay. The control cultures of granulosa cells secreted small amounts of T (700 +/- 317 pg/culture: mean +/-SE) during the first 2 days in vitro, and the addition of LH/FSH, Bu2cAMP, PGF2alpha, or PGE2 did not significantly stimulate T production. After 2 days in vitro, very little T (greater than 200 pg/culture) was produced by control and prostaglandin-treated granulosa cells, whereas those incubated with LH/FSH and Bu2cAMP maintained their initial T production rates. Theca control cultures produced 3 +/- 0.4 ng of T (mean +/- SE) during the first 2 days in 13.6-fold by LH/FSH, 3.6-fold by Bu2cAMP, and 3-fold by PGF2alpha and PGE2- T was not detected in theca cultures after 2 days except in those treated with LH/FSH or Bu2cAMP, which produced 1.5 +/- 0.5 and 1.6 +/- 0.3 ng of T, respectively, at 4 days (mean +/- SE). These results suggest that under the present conditions, pieces of rabbit thecal tissue have a greater capacity to produce T de novo than do isolated granulosa cells, and indicate that T production is transiently stimulated by LH/FSH, Bu2cAMP, PGE2alpha, and PGE2.     

Prostaglandin F2 alpha-induced calcium transient in ovine large luteal cells: II. Modulation of the transient and resting cytosolic free calcium alters progesterone secretion

A previous study demonstrated that prostaglandin F2 alpha (PGF2 alpha) stimulates a transient increase in cytosolic free Ca2+ levels [( Ca2+]i) in ovine large luteal cells. In the present study, the magnitude of the PGF2 alpha (0.5 microM)-induced calcium transient in Hanks' medium (87 +/- 2 nM increase above resting levels) was reduced (P less than 0.05) but not completely eliminated in fura-2 loaded large luteal cells incubated in Ca2(+)-free or phosphate- and carbonate-free medium (10 +/- 1 nM, 32 +/- 6 nM, above resting levels; respectively). Preincubation for 2 min with 1 mM LaCl3 (calcium antagonist) eliminated the PGF2 alpha-induced calcium transient. The inhibitory effect of PGF2 alpha on secretion of progesterone was reduced in Ca2(+)-free medium or medium plus LaCl3. Resting [Ca2+]i levels and basal secretion of progesterone were both reduced (P less than 0.05) in large cells incubated in Ca2(+)-free medium (27 +/- 4 nM; 70 +/- 6% control, respectively) or with 5 microM 5,5'-dimethyl bis-(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (40 +/- 2 nM; 49 +/- 1% control; respectively). In addition, secretion of progesterone was inhibited (P less than 0.05) by conditions that increased (P less than 0.05) [Ca2+]i; that is LaCl3 ([Ca2+]i, 120 +/- 17 nM; progesterone, 82 +/- 8% control) and PGF2 alpha ([Ca2+]i, 102 +/- 10 nM; progesterone, 82 +/- 3% control). In small luteal cells, resting [Ca2+]i levels and secretion of progesterone were reduced by incubation in Ca2(+)-free Hanks ([Ca2+]i, 28 +/- 2 nM; progesterone, 71 +/- 6% control), however, neither LaCl3 nor PGF2 alpha increased [Ca2+]i levels or inhibited secretion of progesterone. The findings presented here provide evidence that extracellular as well as intracellular calcium contribute to the PGF2 alpha-induced [Ca2+]i transient in large cells. Furthermore, whereas an adequate level of [Ca2+]i is required to support progesterone production in both small and large cells, optimal progesterone production in large cells depends upon an appropriate window of [Ca2+]i.     

Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles.

The present studies examined the inhibitory effect of mid-cycle and preovulatory bovine follicular fluid (bFF) on the secretion of progesterone and prostaglandin F 2 alpha (PGF 2 alpha) by bovine theca and granulosa cells cultured in tissue culture Medium 199. The inhibitory effect of bFF on the secretion of PGF 2 alpha by incubated and cultured hemipituitary glands of male rats was also studied. Addition of 5, 10 or 20% charcoal-extracted, mid-cycle bFF to the culture medium caused a twofold decrease in the accumulation of both progesterone and PGF 2 alpha by the cultured granulosa cells (P less than 0.01). In contrast, when preovulatory bFF was used, there was no inhibitory effect on secretion of progesterone and PGF 2 alpha. The addition of 10% mid-cycle bFF to the culture medium caused a two- to fourfold decrease in the accumulation of both PGF 2 alpha and progesterone by the cultured theca (p less than 0.008). However, in the presence of 1 microgram LH/ml, the inhibitory effect of mid-cycle bFF on progesterone and PGF 2 alpha secretion was abolished. The secretion of PGF 2 alpha was significantly (p less than 0.03) decreased in male rat hemipituitary glands after 5 h of incubation or 18 h of culture. These findings suggest that bFF from mid-cycle follicles inhibits prostaglandin synthetase as well as luteinization. The inhibition disappeared with bFF from preovulatory follicles.     

Alteration of prostaglandin production and agonist responsiveness by n-6 polyunsaturated fatty acids in endometrial cells from late-gestation ewes

We investigated the effect of n-6 polyunsaturated fatty acids (PUFAs) on prostaglandin (PG) production by the uterus. A mixed population of endometrial cells (epthelium and stroma) from late-gestation ewes were cultured in defined medium containing linoleic acid (LA, 18:2, n-6), gamma-linolenic acid (GLA, 18:3, n-6) or arachidonic acid (AA, 20:4, n-6) in concentrations of 0 (control), 20 or 100 microM. After 45 h in test medium with or without added PUFAs, cells were challenged with control medium (CM), oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 micro g/ml) or dexamethasone (DEX, 5 microM) for 22 h in the continued presence of the same concentration of PUFA and the medium was collected for measurement of PGF(2alpha) and PGE(2). Supplementation with LA inhibited the production of PGF(2alpha) but did not alter PGE(2), whereas GLA and AA increased production of both PGs. All PUFA supplements thus increased the ratio of PGE(2) to PGF(2alpha) (E:F ratio) two- to threefold. In control cells, OT and LPS challenges stimulated the production of PGF(2alpha) and PGE(2). In all challenge groups, the concentrations of PGF(2alpha) in response to PUFAs followed the same pattern - LA相似文献   

Prostaglandin F2 alpha stimulates cAMP phosphodiesterase via protein kinase C in cultured human granulosa cells.

To investigate the involvement of cyclic AMP phosphodiesterase in the antigonadotrophic actions of prostaglandin F2 alpha (PGF2 alpha), human granulosa cells were cultured in serum-supplemented medium for 72 h, treated for 30 min with cloprostenol or phorbol myristate acetate (PMA) and then exposed to human chorionic gonadotrophin (hCG) +/- isobutyl-methylxanthine (IBMX) for a further 4 h. In the absence of IBMX, cloprostenol and PMA inhibited hCG-stimulated progesterone production. However, in the presence of 0.5 mM IBMX, inhibition of phosphodiesterase prevented these antigonadotrophic effects. Phosphodiesterase activity was assessed by a novel direct assay performed on intact cells after 3 days of culture. PGF2 alpha, cloprostenol and 4 beta-PMA all enhanced cAMP degradation whilst an inactive phorbol ester (4 alpha-PMA) had no effect. Down-regulation of protein kinase C by 4 beta-PMA pre-treatment prevented the subsequent stimulation of phosphodiesterase activity by both cloprostenol and 4 beta-PMA. We conclude that the antigonadotrophic actions of PGF2 alpha in cultured human granulosa cells involve a stimulation of cAMP phosphodiesterase mediated via protein kinase C.     

Mechanism of the rapid antigonadotropic action of prostaglandins in cultured luteal cells

A reproducible method for dissociation and culture of rat luteal cells is described. The concentration of LH required to produce half-maximal stimulation of progesterone secretion was 50 ng/ml. The effects of prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) on basal and luteinizing hormone (LH)-stimulated progesterone production were examined. Both prostaglandins stimulated basal progesterone production but PGE(2) was about twice as active, showing a 2-fold maximal stimulation at 0.75 muM. When either prostaglandin was incubated simultaneously with LH, a dose-dependent inhibition of progesterone secretion occurred; PGF(2alpha) was 4 times more active than PGE(2), showing 50% inhibition at a concentration of 40 x nM. Thus, both prostaglandins are more active as antagonists than as agonists of LH with respect to progesterone secretion. PGF(2alpha) also inhibited LH-stimulated adenylate cyclase activity and cyclic AMP accumulation. The block in progesterone secretion was reversed by addition of dibutyryl cyclic AMP (1 mM) but not by theophylline (5 mM) alone. These data and the finding that PGF(2alpha) did not affect the specific binding activity of the LH receptor in intact luteal cells indicate that the rapid action of prostaglandins in luteal cells is due to a block of LH-dependent production of cyclic AMP which results in a decrease in progesterone secretion.     

Prostaglandin receptors and role of G protein-activated pathways on corpora lutea of pseudopregnant rabbit in vitro

Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.     

Lipocortin output by human endometrium in vitro

Lipocortin was found to be secreted by human endometrium incubated for 1-2 days under organ culture conditions. Levels of lipocortin in the culture medium, measured by RIA, were increased by dexamethasone (10(-8)-10(-6) M) and decreased by progesterone (10(-8)-10(-6) M). Both steroids, however, decreased prostaglandin F2 alpha (PGF2 alpha) output, also estimated by RIA. These results suggest that lipocortin does not mediate the inhibition of PGF2 alpha production evoked by progesterone. Dexamethasone may inhibit PGF2 alpha production by mechanisms involved in the inhibition by progesterone in addition to those mediated by an elevation in the levels of lipocortin, a phospholipase inhibitor.     

Chronic regulation of ovarian oxytocin and progesterone release by prostaglandins: opposite effects in bovine granulosa and early luteal cells

Oxytocin is synthesized in the granulosa-derived large cells of the ruminant corpus luteum from a gene which is dramatically up-regulated in the first few days after ovulation. In this work, the regulation of granulosa and luteal cells by prostaglandins and insulin (or insulin-like growth factor-I; IGF-I) has been explored by comparing their effects on oxytocin and progesterone production in cell culture. In granulosa cells, chronic exposure to insulin (17 nmol/l) stimulated luteinization as indicated by increased release of oxytocin and progesterone. Prostaglandin F2 alpha (PGF2 alpha) alone had little effect, but synergized with insulin (or IGF-I) to increase the release of both these hormones. In direct contrast, insulin-stimulated oxytocin production by luteal cells was inhibited by PGF2 alpha. The half-maximal dose (EC50) for PGF2 alpha action in both cell preparations was similar (10-100 nmol/l). Dose-response studies revealed that PGF2 alpha increased the potency of insulin in granulosa cells (EC50 for insulin-stimulation of oxytocin release reduced from 141 to 13 nmol/l by 1 mumol PGF2 alpha/l), but not in luteal cells. Insulin-stimulated oxytocin release from granulosa cells was also synergistically increased by PGE1, PGE2 and forskolin, suggesting this effect to be mediated by adenylate cyclase-coupled PGE receptors. The results reveal that the effects of prostaglandins on oxytocin release are dependent on both the developmental stage of the target tissue and on the presence of other regulators of cellular differentiation. Moreover, they suggest that the increase in responsiveness to insulin and IGF-I, which appears to accompany luteinization in the cow, may be an effect of prostaglandins produced locally during the peri-ovulatory period.     

The effect of LH/FSH, dibutyryl cyclic AMP, and prostaglandins on the production of estrogens by rabbit granulosa cells in vitro.

The purpose of this study was to establish the effects of trophic hormones on the production of estrogens by rabbit granulosa cells. A pure population of these cells was isolated from preovulatory follicles (1-15 mm in diameter) of estrous rabbits, and cultured for 6 days with either one or a combination of the following hormones: androstenedione, Pergonal (LH/FSH), dibutyryl cyclic AMP (Bu2cAMP), prostaglandin F2alpha (PGF2alpha), or prostaglandin E2 (PGE2). The medium was collected every 2 days and progesterone (P), estrone (E1) and estradiol-17 beta (E2beta) were measured by radioimmunoassay. Granulosa cells cultured as controls (i.e., without exogenous trophic hormones) secreted P spontaneously and its secretion was stimulated 100 to 1,000 fold with LH/FSH and Bu2cAMP, but not with PGF2alpha or PGE2. Androstenedione, either alone or with trophic hormones had no apparent effect on the cytology of the granulosa cells or their ability to secrete P. In the absence of exogenous androstenedione, the cultures produced very small amounts of E1 or E2beta (smaller than 100 pg/ml), either spontaneously or in response to LH/FSH, B12cAMP, PGF2alpha, or PGE2. Incubating granulosa cells with exogenous androstenedione (1 mug/ml) resulted in a 30- to 150-fold increase in E2beta production, which was stimulated an additional 3- to 5-fold with LH/FSH and Bu2cAMP, but not with PGF2alpha or PGE2. In most cultures, E2beta production was restricted to the first 2 days in vitro. Bu2cAMP, however, maintained E2beta production at relatively high levels throughout the duration of the experiment, but there was a progressive decrease in its production. The production of E1 was only 5 percent of E2beta, but the pattern of secretion was similar for both estrogens. These results suggest that cyclic AMP could have a role in regulating the synthesis of estrogens by rabbit granulosa cells.     

Regulation, action and transport of prostaglandins during the periovulatory period in cattle

Follicular production of prostaglandins (PGs) is essential for mammalian ovulation, but the factors that mediate production and the cell-specific action(s) of PGE and PGF2alpha during the ovulatory cascade remain largely unknown. The aims of these experiments were: (1) to investigate the potential role of oxytocin (OT) in ovulatory PG production, (2) to determine cellular and temporal patterns of expression of mRNA for specific PG receptors during the periovulatory interval, (3) to determine cell-specific effects of PGE2 on progesterone secretion, and (4) to investigate the potential for an active transport mechanism that may regulate the effect of PGs during the ovulatory cascade, using cattle as the animal model. Heifers were treated sequentially with PGF2alpha and GnRH to induce luteal regression, a follicular phase and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). In experiment 1, OT increased the secretion of PGE and PGF2alpha by granulosa cells collected from preovulatory follicles (before the LH/FSH surge) and OT production by pieces of follicle wall from periovulatory follicles (after the LH/FSH surge) was regulated by progesterone acting through the progesterone receptor. In experiment 2, levels of mRNA for the PGF2alpha receptor and three PGE receptor subtypes were determined by semi-quantitative RT-PCR in theca interna and granulosa cells from pre- and periovulatory follicles collected at 0, 6, 12, 18 and 24 h post-GnRH. Time- and cell-specific patterns of change in mRNA for PG receptors were observed, suggesting multiple effects of both PGE and PGF2alpha in both theca interna and granulosa cells throughout the ovulatory cascade. Cell-specificity of PG action was confirmed in experiment 3; PGE2 increased the secretion of progesterone by theca interna but not granulosa cells collected from late periovulatory follicles. The results of experiment 4 revealed the expression of mRNA for the bovine PG transporter in theca interna and granulosa cells and its regulation during the periovulatory period, thus revealing the presence of a potential transport mechanism that could regulate cellular distribution of PGs throughout the ovulatory cascade. Taken together, these results provide new insight into mechanisms that regulate the production, distribution and site of action of PGE and PGF2alpha during the ovulatory cascade.     

Effects of oxygen, calcium ionophore, and arachidonic acid on prostaglandin production by monolayer cultures of mixed cells and endothelial cells from rat fetal lungs

Prostaglandins (PGs) synthesized by fetal and neonatal lungs play pivotal roles in pulmonary physiology, especially during the transition from uterine to independent life. One regulator of prostaglandin synthesis at this time may be oxygen. We examined the effects of 1% O2, 21% O2 and 50% O2 in 5% CO2, balance N2 (PO2 values in medium = 30 +/- 4, 142 +/- 4, and 260 +/- 3 mm Hg, respectively), on prostaglandin production from monolayer cultures of mixed or endothelial cells prepared from day 20 gestation rat fetal lungs. Cells were untreated or stimulated to produce prostaglandins by the addition of the calcium ionophore, A23187 (10(-5) M), or the prostaglandin precursor, arachidonic acid (AA, 1 microgram/ml). Prostaglandins 6-keto F1 alpha (6KF, the hydrolysis metabolite of prostacyclin, PGI2), E2, F2 alpha and 13,14-dihydro-15-keto PGF2 alpha (FM, the enzymatic metabolite of PGF2 alpha) were measured by radioimmunoassay. The basal release of 6KF from mixed cells into serum-free medium was approximately 2 ng/10(6) cells/3 days. The levels of 6KF were 10-fold greater than those of the other prostaglandins. Basal endothelial cell release of 6 KF was 30 ng/10(6) cells/3 days, and this was 15- to 100-fold greater than that of the other prostaglandins measured. In mixed cells, oxygen treatment for 3 days had no effect upon the basal release of any prostaglandin, nor was there any effect of oxygen upon the basal 6KF or PGE2 production in endothelial cells. However, both PGF2 alpha and PGFM production by endothelial cells was decreased (p less than 0.05) in 50% O2 compared to 1% O2. Both A23187 and AA enhanced prostaglandin release from mixed and endothelial cells. Ionophore-stimulated 6KF net production in mixed cells was greater in 21% O2 than in 1% O2 (p less than 0.05). Calcium ionophore stimulated the net production of 6KF and PGE2 in endothelial cells in 21% O2 versus 1% O2 (p less than 0.05), and AA enhanced the net production of 6KF, PGE2 and PGF2 alpha in endothelial cells in 21% O2 versus 1% O2. We conclude that rat fetal pulmonary cells produce prostaglandins from endogenous and exogenous substrates, that prostaglandin production is sensitive to Ca2+-mobilizing agents, and that the production of the vasodilators PGI2 and PGE2 increases in the presence of 21% O2 and a stimulating factor.     

Human alveolar macrophages: a lesion in arachidonic acid metabolism in cigarette smokers

Human pulmonary macrophages (PAM) recovered from young cigarette smokers and from age- and sex-matched subjects who had never smoked were used to investigate arachidonic acid metabolism. The uptake of radiolabeled arachidonic acid by PAM obtained from smokers and nonsmokers was not significantly different. The phagocytic indexes of the smoker and nonsmoker macrophages were 17.4 X 3.9% and 18.5 +/- 5.0%, respectively. Both smoker and nonsmoker macrophages produced prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and prostaglandin F2 alpha (PGF2 alpha). A significant decrease in PGE2 and TXB2 synthesis but not in PGF2 alpha synthesis by the smoker PAM compared PAM compared with the nonsmoker PAM was observed using 2 different assays to measure prostaglandin production. Nonsmoker macrophages produced 644 +/- 128,239 +/- 53, and 29 +/- 12 ng/1.5 X 10(6) cells of PGE2, TXB2, and PGF2 alpha, respectively, whereas smoker macrophages synthesized 168 +/- 20, 41 +/- 3, and 17 +/- 1 ng/1.5 X 10(6) cells, as measured by radioimmunoassay. Similar differences were observed using isotopic assays. A cigarette-smoke-induced lesion in phospholipid hydrolysis or the mechanism regulating phospholipid hydrolysis seem most consistent with these findings.     

In vitro effects of ovarian steroids on prostaglandin F2 alpha output by human endometrium and endometrial epithelial cells

Correlations of the rise in prostaglandin F2 alpha (PGF2 alpha) levels in human endometrium during the menstrual cycle with changes in plasma concentrations of ovarian steroids have suggested that progesterone (P) priming of the endometrium is necessary for the stimulation of PGF2 alpha production by estradiol (E2). However, despite the absence of significant levels of P in plasma during the follicular phase of the cycle, PGF2 alpha output by epithelial cell cultures derived from proliferative endometrium was stimulated in vitro by 10(-8) M E2 at least as well as PGF2 alpha output from secretory endometrium. In addition, basal PGF2 alpha output by proliferative endometrium under organ culture conditions was significantly greater than that by secretory endometrium. Concurrent addition of P counteracted the effects of E2 in these in vitro systems. P (10(-7) M) plus E2 (10(-8) M) resulted in PGF2 alpha output as low or lower than those of control incubations of secretory and proliferative endometrium. In the epithelial cell cultures, significant net stimulation by 10(-8) M E2 occurred in the presence of 10(-6) M P, a noteworthy finding since the rise in endometrial PGF2 alpha during the luteal phase takes place when the tissue is exposed to both E2 and P. P lowered the basal output of PGF2 alpha by endometrium in organ culture, but not by epithelial cells in monolayer cultures. E2 appears to stimulate PGF2 alpha output by increasing synthesis rather than diminishing metabolism, since exogenous [3H]PGF2 alpha was metabolized to an equivalent extent by fragments of secretory endometrium or glandular epithelial cells regardless of whether E2 was added to the incubation medium. The results from this study confirm that only secretory endometrium responds to E2 in vitro by significantly increasing PGF2 alpha output. The lack of response by proliferative endometrium, when contrasted with the marked responsiveness of epithelial cells derived from this tissue, suggests that an inhibitory influence is removed during the isolation or subsequent culture of endometrial glands.     

Regulation of eicosanoid production in rabbit colon by interleukin-1

Prostaglandins and thromboxanes are increased in human and experimental colitis, but the factors that regulate this enhanced production are unclear. The present studies evaluate the effects of the monokines, interleukin-1 alpha and beta on eicosanoid production in rabbit colon. In tissue incubations the peak dose response of eicosanoid release to human recombinant interleukin-1 is 50 ng/ml. Interleukin-1 alpha increases prostaglandin E2 (PGE2) by 4.5 +/- 1.9 ng/g tissue, 6-keto PGF1 alpha by 6.2 +/- 2.7 ng/g, and thromboxane B2 by 2.1 +/- 0.3 ng/g compared to placebo. In isolated rabbit colons perfused with Krebs' solution, 10-h infusion of interleukin-1 alpha (50 ng/ml) progressively increases production of PGE2, 6-keto PGF1 alpha, and thromboxane B2. Bolus injections of bradykinin increase production of PGE2, but not 6-keto PGF1 alpha and thromboxane B2, and these responses are markedly augmented by interleukin-1 alpha: at 10 h bradykinin-stimulated PGE2 production is 518 +/- 104 vs. 95 +/- 18 ng/5 min (p less than 0.005), 6-keto PGF1 alpha is 172 +/- 88 vs. 8 +/- 2 ng/5 min (p less than 0.02), and thromboxane B2 is 60 +/- 14 vs. 13 +/- 4 ng/5 min (p less than 0.02) for interleukin-treated colons vs. placebo-treated colons, respectively. The response is greater with interleukin-1 alpha than interleukin-1 beta. This study demonstrates that interleukin-1 stimulates prostaglandin and thromboxane production in normal colon tissue. These data are consistent with the concept that interleukin-1 production by inflammatory cells may augment prostaglandin and thromboxane production in colitis.     

Paracrine regulation of insulin-like growth factor I (IGF-I) an IGF-II on prostaglandins F2alpha and E2 synthesis by human corpus luteum in vitro: a possible balance of luteotropic and luteolytic effects.

The existence of a complete intraovarian insulin-like growth factor (IGF) system replete with ligands, receptors, and binding proteins has been demonstrated as well as the ability of IGF-I to positively affect steroidogenesis in human granulosa cells. Furthermore, we recently showed that IGF-I and IGF-II stimulate progesterone secretion by human luteal cells. As the PGs, PGE2 and PGF2alpha, are classically known to have luteotropic and luteolytic effects, we wanted to determine whether the IGFs could affect the human luteal phase by influencing the PG system. For this reason, human luteal cells were cultured for different times (12, 24, and 48 h) with IGF-I, IGF-II (10-100 ng/mL), and GH (100 ng/mL), and both PGs were assayed in the medium culture. We found that both IGF-I and IGF-II were able to stimulate PGE2 synthesis in a time- and dose-dependent way, whereas they both inhibited PGF2alpha production. GH, too, significantly reduced PGF2alpha synthesis; this effect was IGF-I mediated because it was reverted by increasing dilutions of an anti-IGF-I antibody. On the contrary, no GH effect was observed on PGE2 production. In conclusion, based on these data and on our previous results, we speculate that IGFs could influence luteal steroidogenesis through PG system.     

Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells

The epithelial cell may contribute to the regulation of pulmonary function during inflammatory diseases of the airways by producing metabolites of arachidonic acid (AA). We have used human tracheal epithelial cells (HTE), grown in serum-free medium, to examine cyclooxygenase metabolism of endogenous AA by these cells. Gas chromatography-negative ion mass spectrometry demonstrated that, regardless of stimulus (buffer, bradykinin, or the calcium ionophore A23187), epithelial cells produce PGE2 and PGF2 alpha but no detectable levels of PGD2, thromboxane B2, 6-keto-PGF1 alpha, or 9 alpha, 11 beta-PGF2. Preincubation of cultures with medium containing 5% human serum led to striking increases in the production of PGE2 and PGF2 alpha, regardless of stimulus. Concomitant with these increases in prostanoids, serum exposure caused a 3.6-fold increase in total cellular arachidonate. Arachidonate levels increased in all phosphoglyceride classes, with the greatest increases in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol. In serum-pretreated cells, PGE2 production was 1.46 +/- 0.12, 4.74 +/- 0.6, and 6.35 +/- 0.93 ng/10(6) cells (mean +/- SEM; n = 7) upon exposure to buffer, 10(-6) M bradykinin, and 1 micrograms/ml A23187, respectively, whereas PGF2 alpha levels were 1.53 +/- 0.22, 4.44 +/- 0.36, and 5.77 +/- 0.78 ng/10(6) cells, respectively. The response of HTE to bradykinin was dose-dependent (10(-8) to 10(-6) M) and was maximal within 5 min. We conclude that cyclooxygenase metabolism of endogenous arachidonate in HTE results in the specific production of PGE2 and PGF2 alpha. HTE in culture retain receptors for bradykinin and can be used to study lipid metabolism independent of other cell types.