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Brattleboro rats with hereditary diabetes insipidus make it possible to investigate effects of the urea concentration on the in vitro and in vivo production of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) by the renal papilla independently of any vasopressin effects. The rates of prostaglandin production in vitro are increasing between 100 and 1030 mmol/l urea and decreasing above 1030 mmol/l. The ratio PGE2/PGF2 alpha remains constant at about 4. Normally hydrated and 24 h water-deprived animals in steady state of urine formation were compared in vivo. Urine osmolality increased from 167 +/- 6 (N = 5) to 1113 +/- 35 (N = 15) mosmol/kg water and papillary urea concentration from 50 +/- 7 to 304 +/- 19 mmol/l after water deprivation. Urinary excretion rates of PGF2 alpha increased from 0.83 +/- 0.12 to 3.80 +/- 0.37 ng/h. The excretion of PGE2 was unaffected. PGE2 + PGF2 alpha excretion rates increased from 1.62 +/- 0.25 to 4.61 +/- 0.42 ng/h. These values are in accordance with values predicted from work with Sprague-Dawley rats. Together with previously published data on Sprague-Dawley rats these results indicate that variations of prostaglandin production in the conscious rat in steady state of urine formation can be accounted for by variations of plasma vasopressin and of papillary urea concentration. Variations in the excretion fraction are due to other causes.

This study was conducted as part of an investigation to evaluate the hypothesis that bacterial toxins (LPS or lipoteichoic acid), acting on macrophage-like uterine decidua to cause increased formation of cytokines, may be involved in the pathogenesis of infection-associated preterm labor. We found that cachectin/tumor necrosis factor-alpha (TNF-alpha) was synthesized and secreted into the culture medium by human decidual cells and explants in response to treatment with LPS. LPS treatment also caused an increase in PGF2 alpha production by decidual cells and explants. In amnion cells in monolayer culture, TNF-alpha stimulated PGE2 formation, and TNF-alpha was cytostatic (inhibited [3H]thymidine incorporation into DNA) but not cytolytic in amnion cells. TNF-alpha was not detectable (less than 0.34 ng/ml) in the amniotic fluid of normal pregnancies at midtrimester or at term before or after the onset of labor (n = 44); but TNF-alpha was present at concentrations between 2.8 and 22.3 ng/ml in amniotic fluids of 4 of 20 pregnancies with intact membranes complicated by preterm labor (less than 34 wk gestational age). LPS was present in 10 of the 20 amniotic fluids of preterm labor pregnancies, including all four in which TNF-alpha was present. Bacteria were identified in only one of the four LPS-positive, TNF-alpha-positive fluids. Cytokine formation in macrophage-like decidua may serve a fundamental role in the pathogenesis of preterm labor, including increased prostaglandin formation and premature rupture of the membranes.

There are several controversies related to labor initiation in term. I therefore tried to analyse the relationship between the onset of labor and the dynamics of catecholamine-concentrations in amniotic fluid using the LC-EC method. Results are as follows. 1. Dopamine levels in amniotic fluid increased with gestational age. 2. Norepinephrine and Dopamine levels were higher in women with uterine contraction than in those without uterine contraction. 3. PGF2 alpha production in amnion increased significantly when it was incubated thirty minutes with CAs compared to that incubated only with Hanks' BSS. The PGE production rate in amnion was five or six times as great as that in PGF2 alpha. In conclusion, it is supposed that CAs in amniotic fluid may play a role in the initiation of uterine contraction.

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.

The purpose of this work was to investigate the effects of gonadotropin on the production capacity of ovarian 13,14-dihydro-prostaglandin F2-alpha (13,14H2-PGF2 alpha) and whether or not this capacity had any relation to the process of ovulation in rat. To induce the first ovulation, immature rats were injected subcutaneously with PMSG (5 IU/rat) at 8:00 at 26 days of age and some of these rats were followed by an intraperitoneal injection of hCG (10 IU/rat) at 57 hrs after PMSG treatment. The 13,14H2-PGF2 alpha production capacity was unchanged as compared with vehicle control until 57 hrs after PMSG treatment. However, the capacity showed a striking increase at 60 hrs after PMSG treatment. A maximal increase of about 7 fold was observed at 9 hrs after hCG injection just before ovulation. The production capacity of the Graafian follicle (GF) and the part (WO-GF) of the whole ovary (WO) from which the GF is removed at 2:00 on day 29 and the capacity of early corpus luteum at 8:00 on day 29 was greater than that of GF and WO-GF at 0:00 on day 29. These results suggest that the 13,14H2-PGF2 alpha production capacity in rat ovary is regulated by gonadotropin and is closely associated with the process of ovulation.

Monkey trabecular meshwork (MTM) cells synthesize a variety of prostaglandins, including large amounts of prostaglandin E2 (PGE2) and smaller amounts of 6-keto-PGF1 alpha and PGF2 alpha. The predominance of PGE2 production by the MTM cells is similar to that observed in human trabecular meshwork cells. In contrast, the relative amounts of 6-keto-PGF1 alpha and PGF2 alpha were reversed compared with the human cells. The MTM cells produced increased amounts of PGE2 in response to treatment with bradykinin, platelet activating factor, and A-23187. Dexamethasone caused a dose-dependent inhibition of PGE2 production with 50% inhibition by 10(-8) M, although this response was variable.

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.

1. The synthesis of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane (TX) B2 by isolated glomeruli, cortical tubules, inner medullary slices and outer medullary slices was measured in salt-depleted (LNa) rats and in salt-depleted rats receiving captopril (LNa-CEI). Animals were studied before and after 4, 9 and 15 days of Na+ depletion. 2. Na+ balance was reached in LNa rats after 4 days. Blood pressure and creatinine clearance remained stable. Serum Na+ decreased from 140 +/- 1 to 126 +/- 1 mmol/l (mean +/- SEM, P less than 0.01). In contrast, LNa-CEI rats were unable to conserve Na+ adequately: fractional excretion of Na+ and natriuresis were constantly greater than in LNa animals. As a consequence, LNa-CEI rats developed severe hyponatraemia, lost weight and their creatinine clearance decreased. 3. The glomerular synthesis of PGE2, PGF2 alpha and 6-keto-PGF1 alpha, but not of TXB2, was significantly increased in LNa rats. In LNa-CEI rats, the synthesis of PGE2 and 6-keto-PGF1 alpha was similar to control values, but PGF2 alpha and TXB2 synthesis was elevated at day 9. In cortical tubules, PGE2 and PGF2 alpha were unaffected by Na+ depletion, but 6-keto-PGF1 alpha and TXB2 were increased and a similar trend was observed in LNa-CEI rats. In outer medulla of LNa rats, a decrease in all the eicosanoids measured was observed at day 4. In LNa-CEI animals, the synthesis of PGE2 and PGF2 alpha, but not of 6-keto-PGF1 alpha and TXB2, was significantly depressed. In inner medulla, Na+ depletion only tended to decrease PGF2 alpha and 6-keto-PGF1 alpha, but in the presence of captopril, the synthesis of all prostanoids was significantly decreased.

The production of prostaglandin F2 alpha in monolayer stromal cell cultures of proliferative human endometrium is enhanced by 10(-7) mol/l estradiol-17 beta or 10(-4) mol/l progesterone. Progesterone in high concentration (10(-4) mol/l) also enhanced the synthesis of prostaglandin E2. Clomiphene citrate reduced this increased prostaglandin production dose dependently. The synthesis of prostaglandin I2 was not influenced either by sex steroids or by clomiphene citrate.

In the present study we examined whether the vitamin D3 metabolites, 25-hydroxyvitamin D and 1-25-dihydroxycholecalciferol affected the production of the prostaglandins PGE2 and PGF2 alpha in human fetal membranes and placenta in vitro. Human amnion, chorion, decidual, and placental cells were maintained in primary monolayer culture. Treatment with the vitamin D3 metabolites resulted in an increase in PGE2 and PGF2 alpha production by amnion, decidua, and placental cells; however, these effects varied with time and were different between tissues. Although there was no significant increase in the production of PGE2 and PGF2 alpha by chorion cells in vitro, there was a significant increase in the production of prostaglandin F metabolites after treatment with the vitamin D3 metabolites. The data suggest that the vitamin D3 metabolites may increase free calcium availability and the conversion of arachidonic acid to the prostaglandins. The data do not, however, exclude the possibility that the vitamin D3 metabolites act at other points of arachidonic acid metabolism. These findings raise the possibility of a paracrine role for the vitamin D3 metabolites in the modulation of prostaglandin production within the human fetal membranes and placenta.

Horseradish peroxidase and bovine lactoperoxidase (EC 1.11.1.7), when incubated aerobically with arachidonate, gave rise to the formation of substances identified by bioassay as prostaglandin F2 alpha (PGF2 alpha)- and prostaglandin E2 (PGE2)-like compounds. Boiling of enzymes, which suppressed their capacity to peroxidize guaiacol, also destroyed their capacity to convert arachidonate into PG-like compounds. The rates of formation of PG-like compounds rapidly declined with time, approaching zero after 10 and 20 min for PGE2 alpha- and PGE2-like compounds, respectively. Addition of more enzyme further promoted the reaction. Horseradish and lacto-peroxidases showed optimum pH values of 9.0 and 10.0, respectively. Both enzymes exhibited apparent Km values of about 5 x 10(-5) M for arachidonate. Some reducing agents such as ascorbic acid, NADH and adrenaline dose-dependently inhibited this reaction. The haem poison, phenylhydrazine, also inhibited, with an IC50 of 1 x 10(-7) M. Indomethacin inhibited only the formation of PGE2-like compounds with an IC50 of about 3 x 10(-6) M. As compared to a standard commercial preparation of horseradish peroxidase, the purified horseradish basic and acidic isoenzymes exhibited a higher activity, towards arachidonate whereas other haemoproteins, possessing peroxidase activity, were less active. TLC and GC-MS analyses performed on the reaction products led to the identification of PGF2 alpha, PGE2 and PG6K1 alpha and other unidentified arachidonate derivatives. At 25 degrees, pH 9.5, horseradish peroxidase, acting on saturating concentration of arachidonate, catalysed the formation of 60 mumol/min/mmole enzyme of PGE2 + PGF2 alpha. This appears to be the first report of the synthesis of prostaglandins catalysed by peroxidases.

In order to investigate the role of pericytes (PCs) at microvascular level, a PC line from rat epididymal fat pads was isolated and its prostanoid production in culture was further examined. PC cultures were characterized morphologically by phase contrast and electron microscopy. PC prostanoid production was compared with that of a smooth muscle cell (SMC) line isolated from bovine aorta. The same PGI2 production magnitude was assayed in PC and SMC cultures, but TXA2 and PGF2 alpha synthesis was 8-10 times higher in the PC line. At 4-day postconfluence, when PC layers started retraction, prostanoid synthesis was significantly lower than at confluence. Histamine and bradykinin (both 100 nM) acted similarly, increasing the PGI2 basal production of PC cultures. The results argue for a possible contractile role of PCs at microvascular level.

Eicosanoid synthesis by alveolar macrophages (AM), harvested from tumor bearing animals, was measured after tumor inoculation in rats treated with or without carrageenan (carra), an immunomodulating agent. After incubation of the cells with [14]C-arachidonic acid and the Ca-ionophore A23187, samples were measured by high pressure liquid chromatography (HPLC). From the HPLC profiles the lypoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and leukotriene-B4 (LTB4) were determined as well as the cyclooxygenase products, prostaglandin (PG)E2, PGF2 alpha and TXB2. After tumor inoculation AM-synthesis of lipoxygenase products tended to increase to values twice those of the base line values, whereas cyclooxygenase products showed subnormal values. In the non treated animals, 10 days after tumor inoculation, statistically significant increases in 12- and 15-HETE, LTB4 and PGE2 were observed when compared with carra treated animals. Later measurements did not show these differences in AM metabolism. AM metabolism was (negatively) correlated with the number of macrophages, which was particularly evident in the correlation with 12-HETE synthesis.

The effects of rat calcitonin gene-related peptide (CGRP) on superfused rat aortic smooth muscle cells in cell culture were investigated. Exposure of the cells for 10 min to CGRP (10(-7) M), with or without pretreatment with pertussis toxin, stimulated the release of cyclic AMP but not of prostacyclin, as judged by radioimmunoassay of its stable metabolite, 6-keto-PGF1 alpha. Pretreatment of the cells with pertussis toxin did not alter the response to CGRP. The direct action of CGRP on smooth muscle cells and on the CGRP-induced formation of cyclic AMP did not appear to depend on the production of prostacyclin in these vascular smooth muscle cells.

Estradiol-17 beta increases the production of prostaglandin F2 alpha (PGF2 alpha) in long term monolayer cell cultures of the human endometrium in a dose dependent manner. Progesterone in pharmacological dosage stimulates the syntheses of PGF2 alpha and of prostaglandin E2 (PGE2). The synthesis of prostaglandin I2 (PGI2) is not influenced by sex steroids in long term monolayer cell cultures of the human endometrium.

Rabbit alveolar macrophages were found to produce extraordinary amounts of prostaglandin E2 and F2 alpha with the stimulation of lipopolysaccharide or lipid A. Exogenous prostaglandin E2 greatly enhanced the lipopolysaccharide action on rabbit alveolar macrophages for the induction of prostaglandin F2 alpha release (3-5 fold), while prostaglandin E2 alone did not cause any effect. The enhancement expressed was especially strong when prostaglandin E2 was administered to the cells simultaneously with lipopolysaccharide. The effect of prostaglandin E2 was observed neither with a nonstimulating dose of lipopolysaccharide nor with a stimulating dose of zymosan. This phenomenon was even more pronounced when prostaglandin I2 was used instead of prostaglandin E2, while no sensitization was demonstrated by prostaglandin F2 alpha. These observations suggest that prostaglandins can modulate the activation of the cyclooxygenase pathway of arachidonate metabolism in the activated macrophages by lipopolysaccharide.

Pregnancy and intrauterine infusion of ovine trophoblast protein one (oTP-1) decrease oxytocin-induced secretion of prostaglandin F2 alpha (PGF) from the uterus. In the present study, effects of oTP-1 and pregnancy on endometrial secretion of PGF were examined in an in vitro perifusion system. In Experiment 1, endometrium from day 14 pregnant and cyclic ewes was perifused sequentially on both the lumenal and myometrial sides with Krebs Ringers Bicorbonate solution (KRB), KRB plus oxytocin (1 IU/ml) and KRB alone. Endometrium from pregnant ewes secreted more PGF from both lumenal and myometrial sides than endometrium from cyclic ewes (P less than 0.05). Oxytocin stimulated secretion of PGF from both sides of endometrium regardless of status. Secretion of PGF was greater from the lumenal surface of endometrium compared to myometrium (P less than 0.05) for pregnant and cyclic ewes. For Experiment 2, endometrium was collected from day 15 cyclic ewes and perifused sequentially with KRB, KRB plus 300 ng/ml of either Bovine Serum Albumin (BSA) or oTP-1, KRB with or without BSA or oTP-1 plus oxytocin (1 IU/ml) and then KRB alone. Oxytocin stimulated greater release of PGF from oTP-1-treated than BSA-treated endometrium. Pretreatment of endometrium with oTP-1 had the same effect on oxytocin-induced PGF secretion as cotreatment with oTP-1 and oxytocin. In Experiment 3, uterine horns of cyclic ewes were catheterized on day 10 of the estrous cycle, and infused with either oTP-1 or day 16 pregnant sheep serum proteins on days 12, 13 and 14. Endometrium was collected on day 15 and perifused sequentially with KRB, KRB plus oxytocin (1 IU/ml) and then KRB alone. Treatment of ewes with oTP-1 attenuated endometrial secretion of PGF in response to oxytocin. Results of this study indicate that: (1) pregnancy stimulates basal secretion of PGF from endometrium and has no effect on oxytocin-induced secretion of PGF in vitro; (2) short-term oTP-1 treatment enhances oxytocin-induced PGF secretion from day 15 cyclic endometrium and (3) long-term oTP-1 treatment in vivo inhibits oxytocin-induced PGF secretion in ewes.

Cortisol is known as a potent inhibitor of phospholipase A2 activity in several tissues. In fibroblast monolayer cell cultures from proliferative human endometrium cortisol alone does not affect the basal PGF2 alpha or PGE2 synthesis. After stimulation of PGF2 alpha production by 10(-7) mol/l estradiol-17 beta increasing concentrations of cortisol up to 10(-5) mol/l dosedependently reduce the PGF2 alpha production. Also the progesterone (10(-4) mol/l) stimulated increase of PGF2 alpha and PGE2 synthesis is inhibited by cortisol (10(-7) mol/l).

Using 14C-labeled arachidonic acid as precursor for in vitro prostaglandin synthesis, the effect of an antacid containing Al (OH)3, Mg(OH)2 and CaCO3 on endogenous prostaglandin synthesis was investigated in antral and duodenal mucosa of healthy volunteers. After three weeks of treatment with a high-dose antacid, there was no detectable change in the total capacity of the mucosa for prostaglandin synthesis, but the prostaglandin profile was markedly altered. The relative amounts of PGE2 and PGF2 alpha synthesized by antral and duodenal mucosa increased at the expense of the prostaglandins A2/B2, thromboxane A2, and prostacyclin. In a short-term study, this change was not observed following a single antacid dose within 1 hr after application. It is concluded that long-term antacid treatment may alter the prostaglandin pattern formed by gastroduodenal mucosa and this may be related to its therapeutic effect.

Fifteen patients with subarachnoid hemorrhage (SAH) were divided into two groups; eight patients exhibiting vasospasm (VS) (VS-group), and another seven patients exhibiting no vasospasm (NS-group) during a period of 2-3 weeks after the occurrence of hemorrhage. The cerebrospinal fluid (CSF) of the patients in the VS-group demonstrated a slight but significant decrease in [Mg++] from 1.15 to 0.95 mM on the 6-8th day after the hemorrhage, whereas CSF in the NS-group showed no significant change in [Mg++]. CSF [Ca++] remained relatively unchanged in both groups. The decrease in CSF [Mg++] in the VS-group was not associated with a decrease in [Mg++] in their blood. Probably, it was due to local derangement of the blood-CSF barrier near the site of the actual hemorrhage. Whether or not such a small decrease in CSF [Mg++] could actually influence the occurrence of vasospasm was examined experimentally in vitro. Using helical strips of the bovine middle cerebral arteries, the changes in muscular tension during isometric contraction were recorded on the polygraph. Generally the vascular contractions induced by High K+ (4 X 10(-2) M), PGF2 alpha (10(-6) M), and Oxy-hemoglobin (Oxy-Hb, 3 X 10(-6) M) were augmented when [Mg++] of the perfusing media was reduced from 1.2 to 0.9 mM, and depressed when [Mg++] was increased to 2.4 mM. In descending order, a greater effect of [Mg++] was seen in Oxy-Hb-, and in High K+-, and PGF2 alpha-induced contractions.(ABSTRACT TRUNCATED AT 250 WORDS)

The primary structure of prostaglandin (PG) F synthetase from bovine lung shows 62% similarity with that of human liver aldehyde reductase (EC 1.1.1.2) (Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu, S., Kagamiyama, H., Nakanishi, S., and Hayaishi, O. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 11-15). We therefore purified human liver aldehyde reductase to homogeneity and compared the immunological and catalytic properties of aldehyde reductase and PGF synthetase. Although both enzymes belong to a group of aldoketoreductases and their molecular weights are essentially identical, aldehyde reductase had no cross-reactivity to anti-PGF synthetase antiserum. Furthermore, there was a difference in the substrate specificity for reduction of PGs between the two enzymes. Aldehyde reductase catalyzed the reduction of PGJ2, delta 12-PGJ2, PGH2, or PGA2, but not that of PGB2, PGD2, or PGE2, whereas PGF synthetase reduced PGD2. The optimum pH, Km value for PGH2, and the turnover number were 6.5, 100 microM, and 3.1 min-1, respectively. The PGH2 9,11-endoperoxide reductase activity of aldehyde reductase was not affected in the presence of a substrate such as p-nitrobenzaldehyde, DL-glyceraldehyde, or 9,10-phenanthrenequinone, suggesting that PGH2 9,11-endoperoxide and other substrates are reduced at different active site(s). The reaction product formed from PGH2 by this enzyme was identified as PGF2 alpha by gas chromatography/mass spectrometry. These results suggest that aldehyde reductase is not exactly identical to PGF synthetase in terms of its immunological property and substrate specificity for PGs, but that this enzyme is also involved in the direct conversion of PGH2 to PGF2 alpha similar to PGF synthetase.

Prostaglandins (PGs) are produced by the corpus luteum (CL) of many domestic and laboratory species and may play a role in CL regulation. The production of PGs by luteal tissue of the rhesus monkey has yet to be clearly elucidated. The production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha by CL from rhesus monkeys and the incubation conditions (time and cell number) that permit assessment of their synthesis were examined. CL (n = 3 per characterization) were surgically removed from nonpregnant monkeys during the mid-luteal phase of the menstrual cycle (approximately 8-10 days after ovulation). Luteal tissue was dissociated and the cells were incubated at varying concentrations for increasing periods of time at 37 degrees C. Subsequent to defining incubation conditions, various exogenous factors were examined for their potential to modify PG production. Indomethacin, calcium ionophore, human chorionic gonadotropin (hCG), estradiol-17 beta (E2), progesterone (P), testosterone (T), dihydrotestosterone (DHT), and 1-4-6 androstatriene-3, 17-dione (ATD) were incubated with luteal cells in increasing doses. PG and P concentrations in the medium were determined by radioimmunoassay. PGs in the medium after 6 h incubation were detectable at all cell concentrations tested (50,000, 100,000, 200,000 cells/tube). Concentrations of PGs and P increased with cell number (p less than 0.05). Luteal cells (50,000 cells/tube) were incubated for times of 0-24 h. Concentrations of P, PGE2, and PGF2 alpha in the medium were relatively low prior to incubation (0 h), increased (p less than 0.05) linearly within the first 6-12 h, and plateaued through the remaining 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandins (PG) are produced by the corpus luteum (CL) of the rhesus monkey and may be involved in luteal regulation. Intracellular calcium has also been implicated as a mediator of luteolysis in domestic and laboratory species; however, its role in primate luteal function has not been investigated. The objectives of this study were to characterize temporal changes in basal and stimulated luteal PG production by CL of rhesus monkeys, and to examine the effects of calcium ionophore (CaI) on basal and gonadotropin-stimulated progesterone (P) production by the CL. CL were collected at various times after the estimated day of the luteinizing hormone (LH) surge: 5 days (early luteal phase, n = 4), 8-10 days (mid-luteal phase, n = 8), and 12-14 days (late luteal phase, n = 5). Dispersed luteal cells were incubated in the absence and presence of CaI, or with human chorionic gonadotropin (hCG) plus CaI at 37 degrees C for 8 h. PG and P concentrations in the medium were measured by radioimmunoassay. PGE2 and 6-keto-PGF1 alpha production decreased (p less than 0.05) from early luteal phase to mid-luteal phase and remained lower (p less than 0.05) during late luteal phase for all treatment groups. PGF2 alpha production decreased (p less than 0.05) from early to mid-luteal phase and rebounded in late luteal phase to the same level (p greater than 0.05) found in early luteal phase. CaI stimulated (p less than 0.05) basal PG production. The degree of stimulation was similar throughout the luteal phase (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The formation of cyclooxygenase products in rabbit and rat ocular and non-ocular tissues in vitro, detected by radio-thin-layer chromatography, was inhibited in a concentration-dependent manner by ebselen (PZ 51), an anti-inflammatory seleno-organic compound which has glutathione peroxidase and anti-oxidant activities. The exception was prostaglandin F2 alpha (PGF2 alpha) formation in the rabbit irisciliary body which was stimulated by ebselen in the concentration range 2-10 microM. These observations were confirmed by gas chromatography-mass spectrometry. The concentration that inhibited 50% of prostaglandin biosynthesis (IC50) in the rabbit iris-ciliary body was 9.3 microM. Ebselen also inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) in rabbit and rat ocular tissues and rabbit platelets. The IC50 in the rabbit cornea was 4 microM, whereas higher concentrations were generally required to achieve similar inhibition in other tissues. The formation of 12-HETE by rabbit spleen, however, was not decreased by ebselen at concentrations that were inhibitory in other tissues.

Concurrent changes in concentrations of a product of the cyclooxygenase (prostaglandin [PG] F2 alpha) and lipoxygenase (leukotriene [LT] B4) routes of metabolism of arachidonic acid were measured by radioimmunoassay within the wall of periovulatory ovine follicles. Increased concentrations of PGF2 alpha were detected within follicles before, during and following the time of ovulation. A significant rise in LTB4 was not observed until after follicular rupture had occurred. Inhibition of synthesis of PGF2 alpha by indomethacin was associated with a complete blockade of ovulation. Nordihydroguaiaretic acid, an inhibitor of 5-lipoxygenase, had no effect on ovulation. Periovulatory administration of either drug did not alter sera profiles of progesterone during subsequent luteal phases. These results reconfirm the importance of the cyclooxygenase system in the mechanism of ovulation. It does not appear that follicular LTB4 is a key component in the processes of ovulation or luteinization in sheep.

Differences in prostaglandin (PG) F2 alpha synthesis and degradation were sought between early luteal endometrium (histology day 15 to 22, n = 6) and late luteal endometrium (histology day 23 to 28, n = 6). In addition, alterations in PGF2 alpha synthesis and degradation in response to 17 beta-estradiol (E2) and progesterone (P) were examined to clarify the mechanism of steroid modulation of endometrial PG production (n = 12). Endometrium was maintained in tissue culture and the concentration of PGF2 alpha and 13,14 dehydro-15 keto F2 alpha (DHKF2 alpha) was determined in spent media by radioimmunoassay. Prostaglandin F2 alpha and DHKF2 alpha output from luteal endometrium exposed to P and E2 + P were both significantly reduced when compared with no addition or E2 treatments. This implies that the modulation of PGF2 alpha output by P in vitro is secondary to altered synthesis. There was an increase in PGF2 alpha output from late luteal endometrium when compared with early luteal endometrium in the P and E2 + P treatments, but DHKF2 alpha remained unchanged. These data imply that the temporal increase in PGF2 alpha output is the result of alterations in both PG synthesis and catabolism.

In homogenates of rat cerebral neocortex prostaglandin D2 (PGD2) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from endogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD2 (4.5-11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD2, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9 alpha,11 beta-PGF2 by a NADPH-dependent 11-ketoreductase as has been reported in other human tissues (liver and lung). PGF2 alpha was determined directly as the n-butylboronate derivative. It became clear that 9 alpha,11 beta-PGF2 was formed in considerably greater amounts than PGF2 alpha and that other metabolites are also formed. These results can account for the low amounts of PGD2 found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9 alpha,11 beta-PGF2 must be considered along with other eicosanoids in pathophysiological situations in brain.

Carprofen, a non-steroidal anti-inflammatory drug (NSAID) known to inhibit prostaglandin synthesis, was given intravenously in five cows at a daily dose of 0.7 mg/kg for five days beginning on day 1 postpartum. Blood samples were collected at various times over a period of six days following the first injection. At this dose, carprofen reached highest plasma values of about 45 micrograms/ml after the fifth injection and was well tolerated by all the cows. During the whole experimental period, mean plasma levels of 15-keto-13, 14-dihydro-prostaglandin F2 alpha, the primary metabolite of PGF2 alpha, were significantly (p less than 0.05) lower in treated than in control animals (28-47% vs 64-101% of pretreatment concentrations). The suppressive effect of carprofen on PGF2 alpha-production occurred immediately after its application and was maximal 3-6 h post injection on the first and on the fifth experimental day (60-80% and 40-85%, respectively). We conclude from our results that carprofen in a single dose of 0.7 mg/kg b.w. effectively suppresses PGF2 alpha-release in the postpartum cow. Whether this effect is beneficial in the treatment of uterine inflammatory processes remains to be determined.

The purpose of these studies was to determine the effect of bacterial endotoxin and tumor necrosis factor (TNF) on prostaglandin (PG) secretion by human decidua. Decidual explants were established from women undergoing elective cesarean sections before the onset of labor. Escherichia Coli endotoxin and purified human recombinant TNF (rh TNF) were incubated with decidual explants. PGF2 alpha and PGE2 biosynthesis was measured by radioimmunoassay. A significant increase in the release of all PGs into the media occurred in response to LPS and TNF. In the setting of an extraamniotic infection, bacterial and host secretory products (TNF) could trigger the onset of labor, activating the decidua to produce PGs.

The production of prostaglandins F2 alpha and 6-keto F1 alpha in vitro by the luminal epithelial and the residual (mainly stomal) cells isolated from the uteri of proestrous rats have been measured. The basic procedure involved culturing the cells overnight, washing, and then incubating for 2 h when the amounts of prostaglandins released into the medium were determined by radioimmunoassay. No significant change occurred when the epithelial cells were incubated in the combined presence of arachidonic acid and progesterone for the short-term culture compared to being incubated with arachidonic acid alone. However, when progesterone was incorporated into the medium for the overnight culture, a significant increase occurred in 6-keto PGF1 alpha production by the epithelial cells, without any change in PGF2 alpha, compared to when the cells were incubated overnight in the absence of progesterone. It is suggested that overnight treatment of the epithelial cells with progesterone increases the amount or activity of prostacyclin synthetase, responsible for converting PGH2 endoperoxide to PGI2. Very low levels of prostaglandin production were found for the residual cells.

Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-1+ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01-100 micrograms/ml), nor thymosin alpha 1 (1-10 micrograms/ml), thymulin (0.001-100 ng/ml), thymopoietin II (10-1000 ng/ml) or TP5 (10-1000 ng/ml) affect PGE2, 6-keto-PGF1 alpha, PGF2 alpha and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones.

Immunohistochemical staining for prostaglandin F2-alpha (PG F2 alpha) was conducted to identify PG F2 alpha synthesizing or binding sites in anoxic rat brains. Anoxia was produced in 22 rats to lower the arterial oxygen tension (PaO2) to 21 +/- 4 mmHg by ventilation with a 95% nitrogen and 5% carbon dioxide gas mixture. In 8 animals anoxia was continued for 30 sec, and in 14 rats for 3 min. Prior to decapitation, 5 animals in the 30-sec anoxia group and 8 rats in the 3-min anoxia group were reoxygenated for 5 min, while the remaining 9 were not. Five-min reoxygenation returned the PaO2 to 106 +/- 7. Three non-reoxygenated and 3 reoxygenated rats, all pretreated with indomethacin, and 5 normal rats served as controls. The brains were snap-frozen. The cryosections were stained by the indirect immunofluorescence method. PG F2 alpha was noted mainly in pial vessels in all normal rats. All reoxygenated rats showed a positive reaction not only in blood vessels, but also in neurons, particularly hippocampal neurons and Purkinje cells. The staining of the above neurons was noted to be less in non-reoxygenated rats. The stronger staining was observed in rats reoxygenated after 3-min anoxia than 30-sec anoxia. The indomethacin-pretreated rats showed almost no increase in staining intensity. The above results indicate that reoxygenation after anoxia results in an increase of PG F2 alpha in neurons of both cerebrum and cerebellum.

Glucocorticoids have been shown to decrease prostaglandin I2 synthesis in human endothelial cells, suggesting the possible involvement of lipocortin in the inhibition of arachidonic acid liberation achieved by phospholipase A2 (De Caterina, R., and Weksler, B. B. (1986) Thromb. Haemostasis 55, 369-374). To test this hypothesis, human endothelial cells labeled with [14C]arachidonic acid were stimulated with thrombin (2 units/ml, 10 min), resulting in the secretion of free arachidonic acid together with various 14C-labeled metabolites, mainly 6-keto-prostaglandin F1 alpha, the stable derivative of prostaglandin I2. Under conditions where prior incubation of cells with dexamethasone reduced by 51% 6-keto-prostaglandin F1 alpha production, phospholipid hydrolysis induced by thrombin remained unaffected. Using three rabbit polyclonal antibodies directed against endonexin I, lipocortin I, and lipocortin II, evidence was obtained for the presence in human endothelial cells of equivalent amounts of lipocortin I and an immunologically unrelated 33-kDa protein, together with lower quantities of 67-kDa calelectrin/calcimedin. These Ca2+- and phospholipid-binding proteins were selectively extracted with [ethylene-bis(oxyethylene-nitrilo)]tetraacetic acid (EGTA) from cell membranes precipitated in the presence of Ca2+, and they displayed an inhibitory activity against pig pancreas phospholipase A2. However, the amounts of the three proteins were not changed by cell treatment with 2.5 microM dexamethasone, as detected upon polyacrylamide gel electrophoresis by silver staining, immunoblotting, or autoradiography following [35S]methionine in vivo labeling. Since the antiphospholipase A2 activity of EGTA extracts was hardly modified, it was concluded that an increased synthesis of lipocortin cannot account for the inhibition of prostaglandin synthesis brought about by dexamethasone, suggesting other biological functions for these proteins.

We have investigated the effects of recombinant interleukin-1 beta (rIL-1 beta) on phospholipase A2 activity (PLA2), PLA2 messenger RNA levels, and eicosanoid production in rabbit chondrocytes. Phospholipase A2 activity increased 5 fold with exposure to 1.6 x 10(-11) M rIL-1 beta for 20 hours. An mRNA specific for an extracellular PLA2 was detected by RNA blot hybridization after treatment of the cells with rIL-1 beta. Hydroxylated derivatives of arachidonic acid, including prostaglandins, leukotriene B4, 5-hydroxyeicosa 6E, 8Z, 11Z, 14Z-tetraenoic acid, 12-hydroxyeicosa 5Z, 8Z, 10E, 14Z-tetraenoic acid and 15-hydroxyeicosa 5Z, 8Z, 11Z, 13E-tetraenoic acid increased in cells after rIL-1 beta treatment.

Using immunohistochemical and electron microscopic methods the presence of serotonin and prostaglandin F2 containing granules in some epithelial cells of rat thymus has been shown. The modulatory role of these hormones in the thymic immunocompetent cells production is discussed.

In order to establish metastatic lesions, 2.5 x 10(6) AH100B cells were injected into the left carotid artery of male Donryu rats. Each metastatic nodule in the liver or kidney, 1 mm or less in diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. About 3 weeks later, each ascites was collected from the rats, while not bloody. Then, cancer cells obtained from each ascites were suspended in Dulbecco's PBS without Ca2+ and Mg2+ (pH 7.2) after washing. Then, 10(6) metastasized or control cancer cells were incubated in 0.1 ml of PBS mentioned above together with 0.1 microCi of (1-14C)-AA at 24 degrees C for 3 min, respectively. After the extraction procedure, AA metabolites formed were separated by means of TLC, and each TLC plate was subjected to autoradiography. In the metastasized cells, PG production ability was generally accelerated and especially in that of PGF2 alpha as compared with that of the control.

Radiotracer studies and radioimmunoassay measurements demonstrate that minced tissues of human decidua produce chiefly thromboxane B2 (TxB2) (70% of total eicosanoids) and small amounts of prostaglandin F2 alpha (PGF2 alpha) (13%) PGD2 (8%), 6-keto-PGF1 alpha (5%) and PGE2 (4%). Inhibition of thromboxane synthesis with a specific inhibitor (OKY-1581: sodium (E)-3-[4(-3-pyridylmethyl)-phenyl]-2-methyl propenoate) increased prostaglandin formation in general, with the main product being PGF2 alpha (38%), a nonenzymic derivative of PGH2. Crude particulate fractions prepared from the same tissue synthesized two major products from [3H]arachidonate, TxB2 and 6-keto-PGF1 alpha (54 and 30%, respectively) and some PGF2 alpha and PGE2 (8-8%). However, in the presence of reduced glutathione (GSH), PGE2 became the main product (81%) (TxB2, 15%; PGF2 alpha, 2%; and 6-keto-PGF1 alpha, 2%). Half-maximal stimulation of PGE2 synthesis occurred at 46 microM GSH. The GSH concentration of tissue samples was found to be 110 +/- 30 microM. We conclude that human first trimester decidua cells possess the key enzymes of prostaglandin and thromboxane synthesis. Apparently, the production of these compounds is controlled by a specific mechanism in the tissue, which keeps PGE and prostacyclin synthesis in a reversibly suppressed state, whereas the formation of thromboxane is relatively stimulated.