Production of leukotrienes and prostaglandins in the rat uterus during peri-implantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC4 and LTB4) and prostaglandins (PGE2 and PGF2 alpha) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC4 or LTB4 remained unaltered on days 1-3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE2 and PGF2 alpha showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: vasodilators and agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation per se.     

Further evidence for role of leukotrienes as mediators of decidualization in the rat

Inhibitors of leukotrienes were utilized to investigate the role of leukotrienes (LTs) in the induction of decidualization in the rat. Alzet osmotic minipumps, filled with either FPL 55712 (FPL, a specific antagonist of peptidoleukotrienes), nordihydroguaiaretic acid (NDGA, an inhibitor of LT synthesis) or in combination with leukotriene C4 (LTC4) and/or prostaglandin E2 (PGE2), were instilled at the ovarian end of uterine horns of day 5 pseudopregnant rats. Intraluminal infusion of FPL or NDGA, for 4 days, induced a dose dependent decrease in the uterine wet weights when compared to that induced by the infusion of their corresponding vehicles (1 microliter/h). Furthermore, simultaneous infusion of LTC4 (10 ng/h) with different doses of FPL (1, 0.5, or 0.25 microgram/h) produced an increase in uterine weights as compared to that produced by FPL alone. Maximum response, however, was noted when LTC4 (10 ng/h) was infused with FPL at a rate of 0.5 microgram/h. The infusion of LTC4 (10 ng/h) or PGE2 (1 microgram/h) with NDGA, at 1 and 5 micrograms/h, could not overcome its inhibitory effect on decidualization. On the contrary, a combination of LTC4 (10 ng/h) and PGE2 (1 microgram/h) along with NDGA (5 micrograms/h) significantly increased the uterine weight to a level that was comparable to that induced by the infusion of the vehicle. To determine if the synthesis of PGs and LTs was inhibited by NDGA, one uterine horn was infused with NDGA (5 micrograms/h) and the other horn with the vehicle. The intrauterine infusion of NDGA for 24 h inhibited the release of PGE2, PGF2 alpha, LTC4 and LTB4 as compared to those released by the vehicle-infused horns. These data suggest that both PGs and LTs are required for the induction and progression of decidualization.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe

Interrelationships between production of progesterone (P4), prostaglandin (PG) E2 and PGF2 alpha, and collagenase by periovulatory ovine follicles and their possible involvements in the ovulatory process were investigated. Follicles were isolated from ovaries at intervals (0 to 24 h) after the initiation of the preovulatory surge of luteinizing hormone (LH). Progesterone and PGs within follicles were determined by radioimmunoassay. Digestion of radioactive collagen during coincubation with tissue homogenates was used to assess the production of a bioactive follicular collagenase(s). Follicular accumulation of PGs and P4 increased at 12 and 16 h, respectively, after the onset of the surge of LH; PGE2 then decreased at 20 h. Collagenolytic activity of follicular tissue increased at 20 h and was maximal at 24 h (during the time of follicular rupture). An inhibitor of synthesis of P4 (isoxazol) or PGs (indomethacin) was injected into the follicular antrum at 8 h. Isoxazol did not prevent the initial rise in PGs, but inhibited synthesis of PGF2 alpha at 16 h and therafter. Isoxazol negated the decline in PGE2 and increase in collagenolysis. Indomethacin did not influence synthesis of P4; however, it suppressed collagenolytic activity of follicular tissue. Ovaries with treated follicles were left in situ and observed for an ovulation point at 30 h. Isoxazol or indomethacin was a potent inhibitor of ovulation. The blockade of ovulation by isoxazol was reversed by systemic administration of P4 or PGF2 alpha, but not by PGE2. Reversal of the blockade by indomethacin was accomplished with PGE2 or PGF2 alpha. Collagenolytic activity of follicular tissue was likewise restored by such treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

The detection of 5-lipoxygenase and cyclo-oxygenase products in sputum of patients with chronic bronchitis and bronchiectasis

Leukotrienes (LTs) and prostanoids (Ps) were detected in sputum of patients with chronic bronchitis and/or bronchiectasis (CB/B) using selective superfusion bioassay and radioimmunoassay (RIA) techniques. Analysis of sputum extracts showed a 4-fold increase in the level of LTB4 compared to the cysteinyl-containing LTs (LTC4/LTD4). The measurement of cyclo-oxygenase products (COPs) indicated relatively greater amounts of the vasodilator prostaglandin E2 (PGE2) and prostacyclin (PGI2) compared to the vasoconstrictor prostaglandin F2 alpha (PGF2 alpha) and thromboxane A2 (TxA2) agents (70:30% of total COPs respectively). The presence of eicosanoids (LTs and Ps) in sputum of patients with CB/B suggest that these biologically active substances may act as mediators of bronchoconstriction and inflammation in these diseases.     

Prostaglandin E and E2 alpha secretion by glandular and stromal cells of the pig endometrium in vitro: effects of estradiol-17 beta, progesterone, and day of pregnancy.

Prostaglandins (PGs) are believed to play important roles in the establishment of pregnancy. Glandular and stromal cells were isolated from pig endometrium on days 11 through 19 of pregnancy and cultured in the presence of estradiol-17 beta (E2) and progesterone (P4) to determine the effect of day of pregnancy and steroids on the secretion of PGE and PGF2 alpha. Estradiol at concentrations between .01 and 1 microM did not affect PGE and PGF2 alpha secretion into the medium by glandular and stromal cells. Progesterone (.1 microM) suppressed (P less than .001) PGE and PGF2 alpha production from both cell types. Glandular cells secreted more (P less than .01) PGF2 alpha than PGE, whereas stromal cells collected on days 11, 12, 13, and 19 secreted more (P less than .05) PGE than PGF2 alpha. Stromal cells isolated from tissues collected on day 13 of pregnancy produced PGs with higher (P less than .01) PGE:PGF2 alpha ratio than those from tissues harvested on other days of pregnancy. Glandular cells isolated from tissues collected on days 13 and 19 and stromal cells isolated from tissue collected on day 13 of pregnancy secreted more (P less than .05) PGE and PGF2 alpha than cells isolated on other days of pregnancy. We conclude that: 1) P4 has a suppressing effect on PG secretion; 2) endometrial glandular and stromal cells each produce a unique profile of PGs; and 3) endometrial cells harvested on different days of pregnancy secrete different amounts of PGE and PGF2 alpha.     

Effects of ischemia/reperfusion on brain tissue prostanoids and leukotrienes in newborn pigs.

We investigated the hypothesis that cerebral prostanoid and peptidoleukotriene (LTs) (LTC4/D4/E4/F4) synthesis are increased during postischemic reperfusion of newborn pig brains. Prostanoids and LTs extracted from brain tissue were determined by RIA in sham-control piglets and at 1h, 3h, or 12h after a 20-min period of total cerebral ischemia. During reperfusion following ischemia, all regional brain tissue (cerebrum, brain stem and cerebellum) prostanoids (6-keto-PGF1 alpha, TXB2, PGE2 and PGF2 alpha) were increased at 1h compared with those in sham-control piglets. Only cerebral and brain stem 6-keto-PGF1 alpha and cerebral TXB2 remained elevated at 3h postischemia and all prostanoids returned to control levels by 12h postischemia. Brain tissue LTs were lower than prostanoids and were not altered 1, 3, or 12h following ischemia. These data indicate that 1) newborn pig brain tissue prostanoids are increased initially, and then returned to control levels at later stages of reperfusion following ischemia; 2) LTs are present in newborn pig brain tissue, but are not increased by ischemia/reperfusion injury and therefore probably do not play a significant role in cerebral ischemia-reperfusion injury.     

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.     

Interrelationship between nitric oxide and prostaglandins in bovine granulosa cells

It is well recognized that prostaglandins of the E (PGE) and F (PGF) series play an important role in ovarian physiology; in addition, nitric oxide (NO) has been recently demonstrated to be an important mediator of granulosa cell function. There is now evidence for a biologic relationship between PGs and the NO biosynthetic pathway. The aim of this study was to investigate the relationship between NO and PGE2 and PGF2alpha in bovine granulosa cells. Granulosa cells collected from small (<5mm) and large (>8mm) follicles were treated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or with indomethacin, an inhibitor of PGs synthesis, and PGE2 and PGF2alpha were quantified; in addition, the effects of PGE2 PGF2alpha and indomethacin on steroidogenesis and NO production were determined. The highest concentration of SNAP inhibited (P < 0.001) PGE2 production in cells from both kinds of follicles, while the lowest dose was effective only in cells from small follicles. The highest concentration of SNAP inhibited and stimulated (P < 0.001) PGF2alpha production in cells from small and large follicles, respectively. Progesterone (P4) production was stimulated by PGE2 and inhibited by PGF2alpha (P < 0.001) in cells from both types of follicles. Estradiol 17beta (E2) secretion was inhibited in cells from small and stimulated in those from large follicles by PGE2 (P < 0.05), while PGF2alpha was stimulatory in cells from both kinds of follicles (P < 0.001). P4 production by cells from small follicles was inhibited and stimulated by those from large follicles by indomethacin (P < 0.001), which also increased E2 output in cells from small follicles (P < 0.001). NO production was inhibited by both PGE2 and PGF2alpha except at the lowest concentration, which was stimulatory (P < 0.001). Indomethacin stimulated (P < 0.001) NO production. Taken together, the present data suggest a cross-talk between NO and PGs biosynthetic pathways, which needs to be further clarified.     

Prostaglandin-induced enhancement of the in vitro anamnestic response.

The ability of various prostaglandins (PGs) to affect the in vitro anamnestic immune response of keyhole limpet hemocyanin (KLH)-primed rabbit popliteal lymph node cells was investigated. Of the four PGs studied (PGA1, PGE2 and PGF2alpha), PGE1 was found to have a stimulatory effect, whereas PGA1, PGE2 and pgf2alpha were ineffective in stimulating or inhibiting the in vitro anamnestic response. Under the conditions studied, a 3.5-fold increase in antibody production was obtained in PGE1-treated, KLH-stimulated cultures. Maximum enhancement was obtained when 0.2 mug of PGE1 were added at the time of culture initiation and were allowed to remain in contact with the lymph node cells for 24 hours.     

Uterine and placental prostaglandins and their modulation of oxytocin sensitivity and contractility in the parturient uterus

The parturient uterus develops a markedly enhanced sensitivity to the uterotonic action of oxytocin (OT). The mechanism leading to this enhanced OT sensitivity is not known. Our previous work suggested that prostaglandins (PGs) may be involved. To define the relationship between OT sensitivity and uterine PG production, we measured uterine sensitivity to OT by a quantitative dose-response procedure in rats on Days 19, 20, 21 and 22 of pregnancy and monitored uterine and placental tissue concentrations of PGF2 alpha and PGE2. In addition, we determined the effects of inhibition of endogenous PG synthesis on OT sensitivity and uterine contractility. We found that both OT sensitivity and spontaneous contractility are positively related to uterine PGF2 alpha production. An abrupt increase in OT sensitivity was observed on Days 21 and 22 of pregnancy. The increase in OT sensitivity was coincidental with the marked increase in PGF2 alpha production in the uterus on Days 21 and 22 of pregnancy. Suppression of in vivo PG synthesis caused a reduction in both spontaneous uterine contractility and OT-induced contractions. Uterine PGE2 concentrations and release were 3-5 times lower than PGF2 alpha. There were no significant fluctuations of uterine PGE2 concentration measured on these last 4 days of gestation. Placental PG levels were also found not to be related to uterine contractility. Placental PGE2 levels were higher than PGF2 alpha and may play a regulatory role in placental perfusion. However, placental PGs did not vary with gestational age.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

Prostaglandin production by corpora lutea of rhesus monkeys: characterization of incubation conditions and examination of putative regulators

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)     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Effect of intrarenal adenosine on urinary excretion of prostaglandins and leukotrienes in the anesthesized dog

Adenosine is a renal vasoconstrictor that plays an important role in mediating renal adaptive responses to decreases in renal perfusion pressure. It is known that adenosine acts on the metabolism of arachidonic acid, but the direct repercussions of adenosine in the production of renal prostaglandins and leukotrienes have not been studied. This study was undertaken to evaluate the effect of the intrarenal infusion of adenosine upon the urinary elimination of arachidonic acid derivatives. Samples of urine were collected with lysine acetylsalicylate and determination of prostaglandins (PGs) and leukotrienes (LTs) was performed by radioimmunoassay of samples previously separated by HPLC. The infusion of adenosine decreases the urinary excretion of 6-keto-PGF1 alpha and TxB2 significantly. There was no significant change in urinary excretion of PGE2 while LTB4 and LTC4 showed a tendency to increase. These results suggest that a fall in the synthesis of PGI2 along with an increase in LTC4, which is a constrictor of mesangial cells, could be responsible for the renal vasoconstriction phase of adenosine. Therefore, it was concluded that adenosine vasoconstriction is mediated through the inhibition of the cyclo-oxygenase pathway, diminishing the synthesis of PG vasodilators.     

Effects of prostaglandins and of leukotriene C4 on the metabolism of labelled glucose in uteri isolated from ovariectomized-diabetic rats. Influences of 17-beta-estradiol and of insulin.

The influences of exogenous PGE1, PGE2, PGF2 alpha, LTC4 and insulin (INS) on glucose oxidation in uterine strips isolated from ovariectomized-diabetic (OVD) and ovariectomized-estrogenized-diabetic (OVED) rats, were studied. The spayed animals were made diabetic by a single injection of streptozotocin (65 mg.kg-1 body weight). The effects of prostaglandins were studied in the presence of indomethacin (INDO) in the incubation medium and the effects of LTC4 in the presence of INDO and nordihydroguaretic acid (NDGA). These procedures were followed in order to avoid the possible influences of endogenous derivatives of arachidonic acid formed by the activity of cyclooxygenase and of lipoxygenases. INDO and NDGA did not modify significantly the formation of 14CO2 from U-14C-glucose in uteri from OVD and from OVED rats. INS (0.5 U.ml-1) augmented significantly labelled glucose metabolism, both in OVD as well as in OVED rats. On the other hand, added PGE1, PGE2, PGF2 alpha or LTC4 failed to alter glucose metabolism in uteri from OVD rats. Only PGE1 was able to increase significantly (p less than 0.05) 14CO2 production from labelled glucose in uterine strips from OVED rats. In OVD rats the stimulatory action of INS on uterine glucose metabolism was significantly enhanced by exogenous PGE1, but not modified by PGE2, by PGF2 alpha or by LTC4. PGE1, PGE2 and LTC4 sensitized uterine strips obtained from OVED rats to the effects of INS. The possible importance of PGE1 in improving uterine glucose metabolism in diabetic animals is discussed.     

In vitro prostaglandin production by bovine corpora lutea destined to be normal or short-lived

Basal and calcium ionophore (CaI)-influenced production of prostaglandins (PGs) by corpora lutea (CL) destined to be normal or short-lived were compared. Ovulation was induced in 24 lactating beef cows with human chorionic gonadotropin (hCG, 1000 IU) administered between 35 and 40 days postpartum. Ten cows received norgestomet implants for 9 days prior to induced ovulation (Normal CL) and 14 served as untreated controls (Subnormal CL). Five cows in each treatment were unilaterally ovariectomized on Day 6 (Day 0 = day of hCG administration) and CL were collected. Blood samples were collected daily through-out the experimental period from cows not ovariectomized. Plasma progesterone (P4) in ovary-intact animals indicated that short-lived CL were induced in 8/8 cows not pretreated with norgestomet, and normal luteal lifespan was observed in 4/5 implanted cows. Dispersed luteal cells were incubated for 8 h with 0, 0.05, 0.5, or 5 microM CaI (A23187). Incubation media were analyzed for P4, PGF2 alpha, 6-keto-PGF1 alpha (PGI), and PGE2. The weight, cell number, and basal or CaI-influenced production of P4 did not differ between Normal CL and Subnormal CL. Basal production of PGF2 alpha, PGI, and PGE2 was higher in Subnormal CL than in Normal CL (p less than 0.05). In response to 0.05 microM CaI, PGF2 alpha was stimulated in Subnormal CL (p less than 0.01), while PGI (p less than 0.05) and PGE2 (p less than 0.1) were increased in Normal CL. Production of PGs was reduced by 5 microM CaI in Subnormal CL (p less than 0.01), but not in Normal CL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)