Concentrations Of PGF2 Alpha Research Articles

Pharmacokinetics and anti-inflammatory effects of flunixin meglumine as a sole agent and in combination with phenylbutazone in exercised Thoroughbred horses.

Flunixin meglumine (FM) and phenylbutazone (PBZ) are potent anti-inflammatory agents and as such their potential to mask injuries that would otherwise keep a horse from training or racing is concerning. A common practice in racetrack medicine in the USA is to administer the two drugs within close proximity (24hours apart) of each other, raising the concern of pharmacokinetic interactions and enhanced anti-inflammatory effects. Describe the pharmacokinetics and effects of PBZ on the clearance of FM when administered in close proximity as well as effects on inflammatory mediators. Two-way randomised balanced crossover experiment. Twelve Thoroughbred exercised horses received 500mg FM IV alone or in combination with 2g of IV PBZ 24hours later. Blood and urine samples were collected prior to and for up to 120hours post-drug administration. Whole blood samples were collected at various times and challenged with lipopolysaccharide or calcium ionophore to induce ex vivo synthesis of eicosanoids. Concentrations of FM, PBZ and eicosanoids were measured using LC-MS/MS and noncompartmental pharmacokinetic analysis performed on concentration data. Flunixin meglumine clearance was significantly increased when horses received PBZ 24hours post-administration (P=.03). No other differences in pharmacokinetic parameters were noted between groups. Thromboxane B2 was significantly suppressed, relative to baseline for 96hours post-FM administration. Subsequent administration of PBZ prolonged the suppression. Prostaglandin E2 was decreased for 24hours following administration of FM with subsequent administration of PBZ prolonging the suppression until 120hours. PGF2alpha concentrations were decreased for up to 168hours post-FM administration. FM administration significantly decreased 15-HETE. Small sample size and lack of a phenylbutazone-only treatment group. Administration of PBZ post-FM administration increased FM clearance. The anti-inflammatory effects of FM appear to be prolonged when PBZ is administered 24hours post-administration.

Seminal Plasma Affects Prostaglandin Synthesis and Angiogenesis in the Porcine Uterus1

Introduction of semen into the female reproductive tract may induce molecular and cellular changes facilitating conception and pregnancy. Because prostaglandins (PGs) and appropriate vascularization of the endometrium are crucial for pregnancy success, the effect of seminal plasma (SP) on PG synthesis and angiogenesis was investigated. Gilts at estrus received an infusion of 100 ml of either SP or PBS (control). Uterine horns were collected on Days 1, 3, 5, and 10 after each treatment. Concentrations of PGE2, PGF2alpha , and PGFM were measured in the uterine lumen and endometrial tissue. Expression of PG synthesis pathway enzymes and angiogenic factors, infiltration of immune cells, and vascular bed development were assessed. One day after SP infusion, the PGE2:PGF2alpha ratio in the uterine lumen was lower than in controls. In endometrial tissue, however, PGE2 levels and the PGE2:PGF2alpha ratio were elevated on Day 10. PG-endoperoxide synthase expression in the endometrium was up-regulated on Day 1 and decreased on Day 5 after SP treatment compared to that in controls. PGF2alpha synthase levels were decreased on Day 5 and 10 in SP-treated animals when compared to controls. SP-induced vascular bed development was apparent shortly before the time corresponding to maternal recognition of pregnancy in the pig. Together, these data indicate that the porcine uterus can be sensitized shortly after SP exposure to evoke prolonged effects on PG synthesis and angiogenesis in the endometrium, persisting over the course of the prereceptive phase. Thus, SP can affect uterine receptivity and embryo-maternal interactions in pigs through locally direct and/or indirect mechanisms.

Relationship between infection, inflammation and premature parturition in mares with experimentally induced placentitis

Ascending placentitis results in premature birth and high foal mortality. By understanding how placentitis induces premature delivery, it may be possible to develop diagnostic markers and to delay premature delivery pharmacologically, thereby decreasing perinatal foal mortality. To identify relationships between bacterial infection, inflammation and premature parturition in mares with experimentally induced placentitis. Experiment 1: Concentrations of allantoic fluid prostaglandins (PGs) F2alpha and E2 were measured in 8 mares after intracervical inoculation with Streptococcus equi ssp. zooepidemicus (at Days 285-291 of gestation) until parturition and compared with controls (n = 4). Experiment 2: mRNA expression of interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha and IL-8 in the chorioallantois from inoculated mares in Experiment 1 were compared with 7 mares that foaled normally. Bacterial inoculation resulted in 7 aborted fetuses and birth of one premature, viable foal. Infection was associated with inflammation of the chorioallantois in the region of the cervical star, isolation of bacteria and high concentrations of PGE2 and PGF2alpha in allantoic fluid obtained within 48 h of delivery (P = 0.04). Chorioallantois from all mares expressed mRNA for IL-8, TNF-alpha, IL-6 and IL-1beta. Experimentally infected mares expressed more mRNA for IL-6 (P = 0.003) and IL-8 (P = 0.009) in the cervical star region and more mRNA for IL-6 (P = 0.004) in tissues from placental horns than control mares. Bacterial placentitis may result in liberation of cytokines from the chorioallantois and prostaglandin formation leading to abortion or birth of a precociously mature foal.

The effect of intravesical acetylsalicylic acid instillation on tissue prostaglandin levels after partial bladder outlet obstruction in rabbits

To examine whether obstruction changes the expression of prostaglandins (PGs) in bladder, intravesical low-dose aspirin could be used as a new route of drug administration, this way of administration influences PGs' expression, and contractile function of the bladder is protected after treatment. Eighteen rabbits were divided into three groups. Sham-operated group (group 1) included 6 rabbits. Twelve rabbits were partially obstructed for 70 days. Six of these 12 rabbits not receiving any treatment constituted obstructed group (group 2). The remaining six rabbits received 2 mg/kg/day aspirin (group 3). One rabbit in each group was evaluated on 1st, 7th, 14th, 28th, 42nd, and 70th days following obstructive surgery. After scarification, the percentage of collagenous area and concentrations of PGE2 and PGF2-alpha were measured. Contractile responses to field stimulation (EFS), carbachol, and potassium chloride (KCl) were determined. Wet tissue PGE2 and PGF2-alpha levels were higher in obstructed group than the other groups. Aspirin decreased the percentage of collagenous area in group 3 compared to the group 2, but this difference was not statistically significant. The strips from aspirin groups resulted in better contractile response to cholinergic stimulation with KCl, but this difference was not statistically significant between the obstructed and aspirin groups. Similarly, carbachol did not elicit significantly greater concentration-dependent contraction in strips from obstructed group compared to those from aspirin groups. Maximum responses to EFS were not significant in aspirin group compared to those from obstructed group. Intravesical aspirin may have protective effect on partially obstructed bladder.

Differential Expression of the Enzymatic System Controlling Synthesis, Metabolism, and Transport of PGF2 Alpha in Human Fetal Membranes1

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Gene transcription of TLR2, TLR4, LPS ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia–pyometra complex

Escherichia coli (E. coli) is the most frequent bacterium isolated in cases of cystic endometrial hyperplasia-pyometra complex, the most frequent endometrial disorder in the bitch. Toll-like receptors (TLRs) play an essential role in the innate immune system. The aim of this study was to compare transcription of genes encoding TLR2, TLR4 and LPS ligands (CD14, MD-2, LBP), prostaglandin synthesis enzymes (COX1, COX2, PGES1 and PGFS), and to compare COX1 and COX2 protein expression and PGE(2) and PGF(2alpha) endometrial content in the endometrium of canine diestrous uteri with (n=7) or without (n=7) pyometra. All cases of pyometra were hyperplastic and E. coli was the only isolated bacteria, while diestrous normal uteri did not present signs of cystic endometrial hyperplasia and were negative for bacteriology. Except for COX1, transcription of all genes was significantly higher in pyometra than in normal endometria. COX1 protein was observed in both normal and pyometra uteri, but COX2 protein was only present in pyometra cases. Endometrial PGE(2) and PGF(2alpha) content were significantly higher in pyometra than in normal diestrous endometria. In conclusion, data obtained in this study provides evidence that pyometra-isolated E. coli induces the up-regulation of TLR2 and TLR4 genes in the canine diestrous endometrium. This up-regulation, which is probably the result of the stimulation by LPS and lipoprotein E. coli constituents, leads to the endometrial up-regulation of PG synthesis genes. This, in turn, results in a higher endometrial concentration of PGE(2) and PGF(2alpha), which may further regulate the local inflammatory response.

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.

Preclinical pharmacology of robenacoxib: a novel selective inhibitor of cyclooxygenase‐2

This manuscript reports the results of preclinical studies in the rat with robenacoxib, a novel selective cyclooxygenase (COX)-2 inhibitor. Robenacoxib selectively inhibited COX-2 in vitro as evidenced from COX-1:COX-2 IC50 ratios of 27:1 in purified enzyme preparations and >967:1 in isolated cell assays. Binding to COX-1 was rapid and readily reversible (dissociation t(1/2) << 1 min), whilst COX-2 binding was slowly reversible (t(1/2) = 25 min). In vivo, robenacoxib inhibited PGE2 production (an index of COX-2 inhibition) in lipopolysaccharide (LPS)-stimulated air pouches (ID50 0.3 mg/kg) and for at least 24 h in zymosan-induced inflammatory exudate (at 2 mg/kg). Robenacoxib was COX-1 sparing, as it inhibited serum TxB2 synthesis ex vivo (an index of COX-1 inhibition) only at very high doses (100 mg/kg but not at 2-30 mg/kg). Robenacoxib inhibited carrageenan-induced paw oedema (ID50 0.40-0.48 mg/kg), LPS-induced fever (ID50 1.1 mg/kg) and Randall-Selitto pain (10 mg/kg). Robenacoxib was highly bound to plasma protein (99.9% at 50 ng/mL in vitro). After intravenous dosing, clearance was 2.4 mL/min/kg and volume of distribution at steady-state was 306 mL/kg. Robenacoxib was preferentially distributed into inflammatory exudate; the AUC for exudate was 2.9 times higher than for blood and the MRT in exudate (15.9 h) was three times longer than in blood (5.3 h). Robenacoxib produced significantly less gastric ulceration and intestinal permeability as compared with the reference nonsteroidal anti-inflammatory drug (NSAID), diclofenac, and did not inhibit PGE2 or 6-keto PGF(1alpha) concentrations in the stomach and ileum at 30 mg/kg. Robenacoxib also had no relevant effects on kidney function at 30 mg/kg. In summary, results of preclinical studies in rats studies suggest that robenacoxib has an attractive pharmacological profile for potential use in the intended target species, cats and dogs.

Phospholipase A2 regulation of bovine endometrial (BEND) cell prostaglandin production

BackgroundProstaglandins (PG), produced by the uterine endometrium, are key regulators of several reproductive events, including estrous cyclicity, implantation, pregnancy maintenance and parturition. Phospholipase A2 (PLA2) catalyzes the release of arachidonic acid from membrane phospholipids, the rate-limiting step in PG biosynthesis. The bovine endometrial (BEND) cell line has served as a model system for investigating regulation of signaling mechanisms involved in uterine PG production but information concerning the specific PLA2 enzymes involved and their role in regulation of this process is limited. The objectives of this investigation were to evaluate the expression and activities of calcium-dependent group IVA (PLA2G4A) and calcium-independent group VI (PLA2G6) enzymes in the regulation of BEND cell PG production.MethodsCells were grown to near-confluence and treated with phorbol 12, 13 dibutyrate (PDBu), interferon-tau (IFNT), the PLA2G4A inhibitor pyrrolidine-1 (PYR-1), the PLA2G6 inhibitor bromoenol lactone (BEL) and combinations of each. Concentrations of PGF2alpha and PGE2 released into the medium were determined. Western blot analysis was performed on cellular protein to determine effects of treatment on expression of PLA2G4A, PLA2G6 and PLA2G4C. PLA2 assays were performed on intact cells by measuring arachidonic acid and linoleic acid release and group-specific PLA2 activity assays were performed on cell lysates.ResultsBEND cells produced about 10-fold more PGE2 than PGF2alpha under resting conditions. Production of both PGs increased significantly in response to PDBu-stimulation. PYR-1 significantly diminished production of both PGs by resting cells and abolished the stimulatory effect of PDBu. BEL stimulated production of both PGs. IFNT reduced both PGE2 and PGF2alpha production by resting cells and diminished PDBu stimulation of PG production. Conversely, IFNT did not significantly reduce BEL stimulation of PG production. Cellular expression of PLA2G4A was enhanced by PDBu and this response was diminished by IFNT. Expression of PLA2G6 was not observed to be affected by treatments and no PLA2G4C expression was observed. Arachidonic acid release from intact cells was significantly increased by PDBu and this effect was attenuated by PYR-1 but not by BEL. Release of linoleic acid from intact cells was stimulated by PDBu and inhibited by BEL but not PYR-1. Group specific PLA2-activity assays demonstrated both PLA2G4A and PLA2G6 activity.ConclusionResults from this study demonstrate that PGE2 and PGF2-alpha production by BEND cells is mediated by the activity and expression of PLA2G4A. Interferon-tau treatment diminished expression of PLA2G4A and PG production. BEND cells were shown to express PLA2G6 but, unlike primary or early passage luminal bovine endometrial cells, stimulation of PLA2G6 activity was not associated with increased PG production.

The Role of the Endometrium in Endocrine Regulation of the Animal Oestrous Cycle

A critical analysis of the results of research in the function of the endometrium was carried out and a view point presented. The role of the endometrium in endocrine regulation of the oestrus cycle can be summarized as follows: 1. The transfer of prostaglandin F(2alpha) (PGF(2alpha)) from the uterus to an ovary, which causes luteolysis, occurs mainly via the lymphatic pathways. 2. The system of retrograde transfer of PGs enables PGF(2alpha) and PGE(2) to reach the myometrium and endometrium with arterial blood at high concentration. In the luteal phase, PGF(2alpha), together with the increasing concentration of progesterone, constricts the arterial vessels of the uterus; in the follicular phase and in early pregnancy, PGE(2) together with oestrogen and embryonic signals, relaxes the arterial vessels. In addition, this system protects the corpus luteum from premature luteolysis during the cycle and luteolysis during early pregnancy. 3. In days 10-12 of the cycle, the blood flow in the uterus decreases by 60-70% in pigs and around 90% in sheep. This causes ischaemia and local hypoxia confirmed by the presence of hypoxia inducible factor and thus remodelling of the endometrium commences. 4. The pulsatile elevations in PGF(2alpha) concentration occurring in the blood flowing out of the uterus during the period of luteolysis and the next few days, do not result from increased PGF(2alpha) synthesis as suggested in numerous studies. They are the effect of excretion of PGF(2alpha) and its metabolites together with lymph and venous blood and tissue fluids in which prostaglandin accumulates.

E-Ring Isoprostane Augments Cholinergic Neurotransmission in Bovine Trachealis via FP Prostanoid Receptors

Isoprostanes are prostaglandin-like molecules that accumulate in oxidative stress and also exert powerful biological effects on a wide variety of tissues. We investigated the effects of several different isoprostanes on contractions evoked by electrical field stimulation (EFS) in bovine trachealis, finding only 15-E2t-IsoP to augment those responses. Many others have shown that isoprostanes act on prostanoid receptors, usually those of the thromboxane-selective prostanoid receptor (TP) subtype, although some describe actions through prostaglandin E2-selective prostanoid receptor (EP) or less frequently through prostaglandin F2alpha-selective prostanoid receptors (FP). We used an extensive panel of highly selective agonists and antagonists of prostanoid receptors to characterize the ones through which 15-E2t-IsoP was acting here. Pretreatment with the FP-selective AL-8810 significantly inhibited the augmentation, whereas TP- and EP-selective blockers did not. On the other hand, the augmentation exerted by 15-E2t-IsoP was mimicked by submicromolar concentrations of the FP-selective agonists PGF2alpha and fluprostenol, as well as by micromolar concentrations of the TP-selective agonist U46619. The concentration-response relationship for exogenously added acetylcholine was not significantly affected by 15-E2t-IsoP, confirming that the effect of the latter on EFS-evoked responses was exerted prejunctionally (i.e., to enhance release of Ach from nerve endings), rather than a direct postjunctional effect via a receptor on the smooth muscle. Finally, we investigated whether the inhibitory (adrenergic) innervation was also modulated by 15-E2t-IsoP, finding EFS-evoked relaxations to be unaffected by the isoprostane. We conclude that 15-E2t-IsoP acts upon an FP receptor on the cholinergic nerve endings, leading to enhanced neurotransmission.

Distinct phospholipase A2 enzymes regulate prostaglandin E2 and F2alpha production by bovine endometrial epithelial cells

BackgroundThe 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.MethodsBovine 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.ResultsConstitutive 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.ConclusionResults 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.

Maternal behavior in pigs

When sows kept under commercial conditions were put into crates in the early 1960s, the neuro-endocrine regulation of the maternal behavior in these domestic animals was disputed. Thus, the study of sow maternal behavior intensified and today a significant body of knowledge has accumulated to support the hormonal regulation of sow maternal behavior. The onset of nest building is associated with a periparturient decline in progesterone, an increase in prolactin and a major rise in plasma concentrations of PGF2α the day before parturition. Some nest building behaviors, such as pawing and gathering straw, have been found to correlate with changes in the levels of progesterone, prolactin and somatostatin. The duration of the birth process correlates negatively with peripheral oxytocin levels. During lactation, the stimuli from the piglets affect the release of several hormones which not only regulate the let down of milk but also sow metabolism and mammary milk production. The sow's nursing behavior ensures an even distribution of milk to her piglets. The piglets suckling behavior, in turn, is mainly a way to communicate their individual nutritional needs.

Positive Association, in Local Release, of Luteal Oxytocin with Endothelin 1 and Prostaglandin F2alpha During Spontaneous Luteolysis in the Cow: A Possible Intermediatory Role for Luteolytic Cascade Within the Corpus Luteum1

Luteolysis is caused by a pulsatile release of prostaglandin F(2alpha) (PGF(2alpha)) from the uterus in ruminants, and a positive feedback between endometrial PGF(2alpha) and luteal oxytocin (OXT) has a physiologic role in the promotion of luteolysis. The bovine corpus luteum (CL) produces vasoactive substances, such as endothelin 1 (EDN1) and angiotensin II (Ang II), that mediate and progress luteolysis. We hypothesized that luteal OXT has an additive function to ensure the CL regression with EDN1 and Ang II, and that it has an active role in the luteolytic cascade in the cow. Thus, the aim of the present study was to observe real-time changes in the local secretion of luteal OXT and to determine its relationship with other local mediators of luteolysis. Microdialysis system (MDS) capillary membranes were implanted surgically into each CL of six cyclic Holstein cows (18 lines total among the six cows) on Day 15 (estrus == Day 0) of the estrous cycle. Simultaneously, catheters were implanted to collect ovarian venous plasma ipsilateral to the CL. Although the basal secretion of OXT by luteal tissue was maintained during the experimental period, the intraluteal PGF(2alpha) secretion gradually increased up to 300% from 24 h after the onset of luteolysis (0 h; time in which progesterone started to decrease). In each MDS line (microenvironment) within the CL, the local releasing profiles of OXT were positively associated with PGF(2alpha) and EDN1 within the CL in all 18 MDS lines implanted in the six CLs (OXT vs. PGF(2alpha), 50.0%; OXT vs. EDN1, 72.2%; P < 0.05). On the other hand, the intraluteal OXT was weakly related to Ang II (OXT vs. Ang II, 27.7%). In the ovarian vein, the peak concentration of PGF(2alpha) increased significantly when the peak of PGF(2alpha) coincided with the peak of OXT after the onset of spontaneous luteolysis (P < 0.05). In conclusion, intraluteal OXT may locally modulate secretion of vasoactive substances, particularly EDN1 and PGF(2alpha) within the CL, and thus might be one of the luteal mediators of spontaneous luteolysis in the cow.

Gene Silencing of Cyclooxygenase-2 mRNA by RNA Interference in Bovine Cumulus-Granulosa Cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.

Role of the prostaglandins in labour and prostaglandin receptor inhibitors in the prevention of preterm labour

Parturition is composed of five separate but integrated physiological events: fetal membrane rupture, cervical dilatation, myometrial contractility, placental separation, and uterine involution. Prostaglandins (PGs) have central roles in each of these events, but the most studied is myometrial contraction. Elevated uterine PGs or the enhanced sensitivity of the myometrium to PGs leads to contractions and labour. The primary regulator of PG synthesis is the mRNA expression of PG H Synthase (PGHS-2 or COX-2). Given the central role of PGs in labour, this enzyme becomes an obvious therapeutic target for the prevention of preterm labour, the major cause of perinatal mortality and morbidity. Unfortunately, even though the non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit PGHS, are usually successful in suppressing preterm labour or prolonging pregnancy in animal and human studies, the NSAIDS have had adverse effects on fetal physiology and development. Therefore, other means to suppress PG synthesis or action to arrest preterm labour need to be investigated. The PGF2alpha receptor, FP, may prove to be a reasonable target for tocolysis. FP mRNA increases in the mouse uterus at preterm birth, whereas PGF2alpha concentrations do not increase, suggesting elevated uterine sensitivity to contractile agonists is one mechanism for preterm labour initiation. New data shows that administration of a specific FP antagonist, Theratechnologies (THG) 113.31, delays preterm birth in mice and sheep with no observable maternal or fetal side effects. Hence antagonizing PG action offers new hope for delaying preterm birth.

Leptin Suppresses Human Chorionic Gonadotropin-Induced Cyclooxygenase-2 Expression and Prostaglandin Production in Cultured Human Granulose Luteal Cells

In a previous study, we demonstrated that high leptin levels at the time of human chorionic gonadotropin (hCG) injection impaired the pregnancy rate for women undergoing in vitro fertilization. In this study we examine leptin's effect on prostaglandin formation and cyclooxygenase (COX) expression induced by hCG in human granulose luteal (GL) cells. Human GL cells were obtained from women undergoing ovarian hyperstimulation. COX expression and microsomal prostaglandin E synthase (mPGES) expression, as well as prostaglandin E(2) (PGE(2)) and prostaglandin F(2alpha) (PGF(2alpha)) production were studied. This was done in both the presence and absence of leptin following hCG stimulation. PGE(2) and PGF(2alpha) were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of COX and mPGES were investigated by using immunocytochemical techniques in addition to Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. HCG and leptin do not affect COX-1 expression. However, leptin blocked COX-2 and mPGES expression induced by hCG. Moreover, while leptin, in various concentrations, did not affect PGE(2) and PGF(2alpha) levels, it inhibited the elevation of PGE(2) and PGF(2alpha) concentrations in response to hCG. The study confirms that the expression of COX-2 is up-regulated by hCG in human GL cells. Leptin suppresses hCG-induced PGE(2) formation through the inhibition of COX-2 and mPGES expression. The preliminary results suggest a potential inhibiting effect of leptin on human GL cells induced by hCG.

Omega-3 fatty acids and decidual cell prostaglandin production in response to the inflammatory cytokine IL-1β

The objective of this study was to determine the effect of omega-3 fatty acids (eicosapentaenoic acid [EPA]; docosahexaenoic acid [DHA]) on prostaglandin production and prostanoid enzyme expression in cultured decidual cells exposed to interleukin-1beta (IL-1beta), a cytokine that plays a major role in inflammation. Decidua was obtained from human placentas of nonlaboring patients at term cesarean delivery (N = 6) and cultured by using standard cell culture techniques. Cells were preincubated in defined media with various concentrations of vehicle, DHA, or EPA for 1 hour. IL-1beta (10 ng/mL) was then added to the media, and experiments were terminated 12 hours after exposure to IL-1beta. Prostaglandin E2 (PGE2) and PGF2alpha concentrations in conditioned media were measured by enzyme-linked immunosorbent assay; cyclooxygenase-1 (COX-1), COX-2, microsomal prostaglandin E synthase (mPGES)-1, mPGES-2, and 15-hydroxy prostaglandin dehydrogenase (PGDH) expression were quantified by real-time polymerase chain reaction and immunoblotting. Groups were compared with the use of Student t test, with significance defined as P < .05. Preincubation with DHA decreased prostaglandin production by up to 80% when compared with controls. DHA decreased both mPGES-1 and -2 messenger RNA expression by approximately 50% (P = .02). Preincubation in DHA or EPA had no effect on COX-1, COX-2, and PGDH messenger RNA or protein expression. Under conditions simulating inflammation, supplementation with omega-3 fatty acids decreases PGE2 and PGF2alpha production in cultured decidual cells. The reduction in prostaglandin production was associated with a decreased expression of mPGES-1 and -2. These findings suggest a mechanism by which omega-3 fatty acid supplementation decreases the incidence of preterm birth in high-risk patients.

Dietary (n-3) Fatty Acids Reduce Plasma F2-Isoprostanes but Not Prostaglandin F2α in Healthy Humans

(n-3) Fatty acids are unsaturated and are therefore easily subject to oxidization; however, they have several beneficial health effects, which include protection against cardiovascular diseases. The aim of this study was to investigate whether (n-3) fatty acids, with a controlled fat quality in the background diet, affect nonenzymatic and enzymatic lipid peroxidation and antioxidant status in humans. A total of 162 men and women in a multicenter study (The KANWU study) were randomly assigned to a diet containing a high proportion of saturated fatty acids or monounsaturated fatty acids (MUFA) for 3 mo. Within each diet group, there was a second random assignment to supplementation with fish-oil capsules [3.6 g (n-3) fatty acids/d] or placebo. Biomarkers of nonenzymatic and enzymatic lipid peroxidation in vivo were determined by measuring 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)) and prostaglandin F(2alpha) (PGF(2alpha)) concentrations in plasma at baseline and after 3 mo. Antioxidant status was determined by measuring plasma antioxidant capacity with an enhanced chemiluminescence assay. The plasma 8-iso-PGF(2alpha) concentration was significantly decreased after 3 mo of supplementation with (n-3) fatty acids (P = 0.015), whereas the PGF(2alpha) concentration was not affected. The antioxidant status was not affected by supplementation of (n-3) fatty acids, but was improved by the background diet with a high proportion of MUFA. We conclude that supplementation with (n-3) fatty acids decreases nonenzymatic free radical-catalyzed isoprostane formation, but does not affect cyclooxygenase-mediated prostaglandin formation.

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.