Induction of luteolysis in mares utilizing a micro-dose of prostaglandin F-2 alpha in the sacral lumbar space

An experiment was conducted to examine the luteolytic effectiveness of using low or micro doses of PGF2α when administered at the BAI HUI acupuncture point, which is frequently used to treat ovarian disturbances in veterinary acupuncture. The results indicate that PGF2α given at a very low micro dose of 0.5 mg (one tenth the conventional recommended dose) administered at the BAI HUI acupuncture point located at the sacral lumbar space is equally effective at inducing luteolysis in mid-luteal phase mares as conventional PGF2α i.m. treatment using a tenfold higher dose. Therefore, based on the results of the present study, we suggest that the BAI HUI sacrai lumbar route somehow provides an extremely efficient pathway for the drug to the ovarian level.

Reproductive parameters of dairy goats submitted to estrus synchronization with prostaglandin F2a associated or not to hCG at estrous onset

Coodernação de Aperfeiçoamento de Pessoal de Nível Superior(CAPES)

Prostaglandin receptors (EP2 and EP4) and angiotensin receptor (AGTR2) mRNA expression increases in the oviducts of Nelore cows submitted to ovarian superstimulation

Many peptides are responsible for the coordination of muscle contraction, secretion and ciliary beating of the oviduct epithelium to allow the transport of gametes and embryos, including vascular endothelial growth factors (VEGF), prostaglandins (PGs), endotelin-1 (ET-1) andangiotensinII(Ang II). The effect of reproductive biotechnologiesusedto improve embryo yield on oviduct gene expression is poorly understood. Thus, the aim of the present study was to evaluate the effect of ovarian superstimulation on the mRNA expression of the genes encoding the major peptides involved in oviduct contraction in bovine. Therefore, Nelore cows were submitted to P-36 (n = 5) or P-36/eCG (n = 5) ovarian superstimulatory protocols and a control group of cows was not submitted to any superstimulatory protocol (n = 5). The relative expression of VEGF (VEGF, Flk1, Flt1), Ang II (AGTR2, ACE1), ET1 (ET1, ECE1) and PG pathway members (PGES, EP2, EP4, COX1, COX2) was analyzed using real time RT-PCR in each of oviduct segment (infundibulum, ampulla and isthmus). All target genes were expressed in the three segments of the bovine oviduct; however, specific genes were regulated by ovarian superstimulation: EP2 and EP4 receptors mRNA was affected by P- 36/eCG protocol, in the ampulla and infundibulum, respectively; and AGTR2 mRNA was up-regulated by both the P-36/eCG and P-36 protocols in the isthmus. The upregulation of EP2, EP4 and AGTR2 expression in the superstimulated cows suggests a suitable effect of FSH and eCG on bovine oviduct physiology, coordinating the contraction in Nelore cows

Febrile response induced by cecal ligation and puncture (CLP) in rats: involvement of prostaglandin E-2 and cytokines

The purpose of the present study was to better understand the events involved in the febrile response induced by cecal ligation and puncture (CLP), a complex infectious process. To this end, we conducted in vivo experiments in rats examining (1) fever development, (2) bacterial number in the infection focus and in blood, (3) peripheral and hypothalamic synthesis of cytokines, (4) hypothalamic and cerebrospinal fluid (CSF) synthesis of prostaglandin E-2 (PGE(2)), (5) the effect of anti-IL-6 antibody on fever, and (6) the effect of celecoxib on fever and hypothalamic synthesis of PGE(2) after CLP induction. We found that CLP promotes fever and animal death depending on the number of punctures. The peak of CLP-induced fever overlapped with the maximal increase in the number of bacteria in the infectious focus and blood, which occurred at 6 and 12 h. The peak of the febrile response also coincided with increased amounts of interleukin (IL)-1 beta, IL-6 and IL-10 in the peritoneal exudate and serum; IL-6 in the hypothalamus and PGE(2) in the CSF and predominantly in the hypothalamus. Moreover, intracerebroventricularly injected anti-IL-6 antibody reduced the febrile response while celecoxib reduced the fever and PGE(2) amount in the hypothalamus induced by CLP. Tumor necrosis factor (TNF)-alpha peaked at 3 h at all sites studied. Conversely, IL-10 concentration decreased in the hypothalamus. These findings show that the peak of CLP-induced fever is accompanied by an increase of bacteria in peritoneal fluid (local infection) and blood; local synthesis of pyrogenic (IL-1 beta, IL-6) and antipyretic (IL-10) cytokines and central production of IL-6 and PGE(2), suggesting that these last are the central mediators of this response.

Prostaglandin treatment at the onset of norgestomet and estradiol-based synchronization protocols did not alter the ovarian follicular dynamics or pregnancy per timed artificial insemination in cyclic Bos indicus heifers

The aim of the present study was to evaluate the effects of the PGF2˛treatment givenat the onset of a synchronization of ovulation protocol using a norgestomet (NORG) earimplant on ovarian follicular dynamics (Experiment 1) and pregnancy per AI (P/AI; Exper-iment 2) in cyclic (CL present) Bos indicus heifers. In Experiment 1, a total of 46 heiferswere presynchronized using two consecutive doses of PGF2˛12 days apart. At first dayof the synchronization protocol the heifers received implants containing 3 mg of NORGand 2 mg of estradiol benzoate (EB). At the same time, heifers were randomly assignedto receive 150 mg of d-cloprostenol (n = 23; PGF2˛) or no additional treatment (n = 23;Control). When the ear implants were removed 8 days later, all heifers received a PGF2˛treatment and 1 mg of EB was given 24 h later. The follicular diameter and interval toovulation were determined by transrectal ultrasonography. No effects of PGF2˛treat-ment on the diameter of the largest follicle present were observed at implant removal(PGF2˛= 9.8 ± 0.4 vs. Control = 10.0 ± 0.3 mm; P = 0.73) or after 24 h (PGF2˛= 11.1 ± 0.4 vs.Control = 11.0 ± 0.4 mm; P = 0.83). No differences in the time of ovulation after ear implantremoval (PGF2˛= 70.8 ± 1.2 vs. Control = 73.3 ± 0.9 h; P = 0.10) or in the ovulation rate(PGF2˛= 87.0 vs. Control = 82.6%; P = 0.64) between treatments were observed. In Experi-ment 2, 280 cyclic heifers were synchronized using the same experimental design describedabove (PGF2˛; n = 143 and Control; n = 137), at random day of the estrous cycle. All heifersreceived 300 IU of equine chorionic gonadotropin (eCG) and 0.5 mg of estradiol cypionate(as ovulatory stimulus) when the NORG ear implants were removed. Timed artificial insem-ination (TAI) was performed 48 h after implant removal and the pregnancy diagnosis wasconducted 30 days later. No effects on the P/AI due to PGF2˛treatment were observed(PGF2˛= 51.7 vs. Control = 57.7%; P = 0.29). In conclusion, PGF2˛treatment at the onset ofNORG-based protocols for the synchronization of ovulation did not alter the ovarian follic-ular responses or the P/AI in cyclic Bos indicus beef heifers synchronized for TAI.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Increases Prostaglandin E2 in Pancreatic Tumors

Prostaglandin E2 (PGE2) is a product of cyclooxygenase (COX) and PGE synthase (PGES) and deactivated by 15-hydroxyprostaglandin dehydrogenase (PGDH). Down-regulation of PGDH contributes to PGE2 accumulation in lung and colon cancers but has not been identified in pancreatic cancer.

Effects of a single administration of prostaglandin F2alpha, or a combination of prostaglandin F2alpha and prostaglandin E2, or placebo on fertility variables in dairy cows 3-5 weeks post partum, a randomized, double-blind clinical trial

BACKGROUND: Delayed uterine involution has negative effects on the fertility of cows; use of prostaglandin F2alpha alone as a single treatment has not been shown to consistently improve fertility. Combined administration of PGF2alpha and PGE2 increased uterine pressure in healthy cows. We hypothesized, that the combination of both prostaglandins would accelerate uterine involution and have, therefore, a positive effect on fertility variables. In commercial dairy farming, the benefit of a single post partum combined prostaglandin treatment should be demonstrated. METHODS: 383 cows from commercial dairy farms were included in this study. Uterine size and secretion were evaluated at treatment 21-35 days post partum and 14 days later. Cows were randomly allocated to one of three treatment groups: PGF2alpha and PGE2, PGF2alpha or placebo. For every animal participating in the study, the following reproduction variables were recorded: Interval from calving to first insemination, days open, number of artificial inseminations (AI) to conception; subsequent treatment of uterus, subsequent treatment of ovaries. Plasma progesterone level at time of treatment was used as a covariable. For continuous measurements, analysis of variance was performed. Fisher's exact test for categorical non-ordered data and exact Kruskal-Wallis test for ordered data were used; pairwise group comparisons with Bonferroni adjustment of significance level were performed. RESULTS: There was no significant difference among treatment groups in uterine size. Furthermore, there was no significant difference among treatments concerning days open, number of AI, and subsequent treatment of uterus and ovaries. Days from calving to first insemination tended to be shorter for cows with low progesterone level given PGF2alpha and PGE2 in combination than for the placebo-group (P = 0.024). CONCLUSION: The results of this study indicate that the administration of PGF2alpha or a combination of PGF2alpha and PGE2 21 to 35 days post partum had no beneficial effect upon measured fertility variables. The exception was a tendency for a shorter interval from calving to first insemination after administration of the combination of PGF2alpha and PGE2, as compared to the placebo group. Further research should be done in herds with reduced fertility and/or an increased incidence of postpartum vaginal discharge.

Prostaglandin E1 testing in heart failure-associated pulmonary hypertension enables transplantation: the PROPHET study

BACKGROUND: Elevated pulmonary vascular resistance (PVR) is relevant to prognosis of congestive heart failure and heart transplantation. Proof of reversibility by pharmacologic testing in potential transplantation candidates is important because it indicates a reduced probability of right ventricular failure or death in the early post-transplant period. This study aimed to clarify the possible extent of acute reversibility of elevated PVR in a large, consecutive cohort of heart transplant candidates. METHODS: This study included 208 consecutive patients (age 52 +/- 10 years, 89% men and 11% women, ejection fraction 21 +/- 9%, Vo2max 12.6 +/- 4.2 ml/kg/min) being evaluated for heart transplantation in 7 transplant centers in Germany and Switzerland. Testing was performed with increasing intravenous doses of prostaglandin E1 (PGE1; average maximum dose 173 +/- 115 ng/kg/min for at least 10 minutes) in 92 patients exhibiting a baseline PVR of > 2.5 Wood units (WU) and/or a transpulmonary gradient (TPG) of > 12 mm Hg. RESULTS: PGE1 testing lowered PVR from 4.1 +/- 2.0 to 2.1 +/- 1.1 WU (p < 0.01), increased cardiac output from 3.8 +/- 1.0 to 5.0 +/- 1.5 liters/min (p < 0.01), and decreased TPG from 14 +/- 4 to 10 +/- 3 mm Hg (p < 0.01), mean pulmonary artery pressure (PAM) from 39 +/- 9 to 29 +/- 9 mm Hg (p < 0.01) and mean pulmonary capillary wedge pressure (PCWP) from 24 +/- 7 to 19 +/- 9 mm Hg (p < 0.01). Mean aortic pressure (MAP) decreased to 85% and systemic vascular resistance (SVR) to 65% of baseline values (p < 0.01). Symptomatic systemic hypotension was not observed. For the whole population the percentage of patients with PVR > 2.5 WU was reduced from 44.2% to 10.5% with PGE1. PVR decreased in each patient; only 2 patients (1%) remained ineligible for listing because of a final PVR of > 4.0 WU. TPG, ejection fraction and male gender were independent predictors of reversibility of PVR. CONCLUSIONS: Elevated PVR in heart transplant candidates is highly reversible and can be normalized during acute pharmacologic testing with PGE1.

Effects of Delta12-prostaglandin J2 on bone regeneration and growth factor expression in rats

OBJECTIVES: Cyclopentenone prostaglandins have been shown to promote osteoblast differentiation in vitro. The aim of this study was to examine in a rat model the effects of local delivery of Delta(12)-prostaglandin J(2) (Delta(12)-PGJ(2)) on new bone formation and growth factor expression in (i) cortical defects and (ii) around titanium implants. MATERIAL AND METHODS: Standardized transcortical defects were prepared bilaterally in the femur of 28 male Wistar rats. Ten microliters of Delta(12)-PGJ(2) at 4 concentrations (10(-9), 10(-7), 10(-5) and 10(-3) mol/l) in a collagen vehicle were delivered inside a half-cylindrical titanium chamber fixed over the defect. Contralateral defects served as vehicle controls. Ten days after surgery, the amount of new bone formation in the cortical defect area was determined by histomorphometry and expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor (IGF)-I/II, bone morphogenetic protein (BMP)-2 and -6 was examined by immunohistochemistry. In an additional six rats, 24 titanium implants were inserted into the femur. Five microliters of carboxymethylcellulose alone (control) or with Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) were delivered into surgically prepared beds prior to implant installation. RESULTS: Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) significantly enhanced new bone formation (33%, P<0.05) compared with control cortical defects. Delivery of Delta(12)-PGJ(2) at 10(-3) mol/l significantly increased PDGF-A and -B and BMP-2 and -6 protein expression (P<0.05) compared with control defects. No significant difference was found in IGF-I/II expression compared with controls. Administration of Delta(12)-PGJ(2) also significantly increased endosteal new bone formation around implants compared with controls. CONCLUSION: Local delivery of Delta(12)-PGJ(2) promoted new bone formation in the cortical defect area and around titanium implants. Enhanced expression of BMP-2 and -6 as well as PDGF-A and -B may be involved in Delta(12)-PGJ(2)-induced new bone formation.

Influence of prostaglandin E2 on parturition in cattle

A double-blinded, randomised, placebo-controlled field study of the influence of prostaglandin E2 (PGE2) on cattle at parturition was carried out. The extent of cervical opening and the intensity of labour were scored before administration of the compound and 10 minutes later; routine birth assistance was then continued by the veterinarian. Successful birth occurred more quickly in the cows treated with PGE2. The extent of cervical opening before the administration of the drug had a significant effect on the time to delivery, but the intensity of labour and a concomitant infusion of calcium did not have significant effects on this period. The less open the cervix before administration of the drug, the more the duration of parturition differed between the two groups, with the placebo group taking longer. A telephone follow-up inquiry found no significant differences between the cows postpartum; there were cases of mastitis and hypocalcaemia in both groups. The incidence of retained fetal membranes and the mortality of the calves were higher in the placebo group, but in neither case was the difference significant.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

CREB mediates prostaglandin F2alpha-induced MUC5AC overexpression.

Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are PGE(2) and PGF(2alpha). PGE(2)-induced mucin production has been well studied, but the effect of PGF(2alpha) on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF(2alpha) on MUC5AC production, we investigated the signal transduction of PGF(2alpha) associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF(2alpha) induces MUC5AC overproduction via a signaling cascade involving protein kinase C, ERK, p90 ribosomal S6 protein kinase, and CREB. The regulation of PGF(2alpha)-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF(2alpha) receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE(2) and other inflammatory mediators, our findings have important clinical implications for the management of airway mucus hypersecretion.

ROLE OF PROSTAGLANDIN E2 IN THE REGULATION OF PANCREATIC STELLATE CELLS HYPER ACTIVITY ASSOCIATED WITH PANCREATIC CANCER

Pancreatic cancer is one of the most lethal type of cancer due to its high metastasis rate and resistance to chemotherapy. Pancreatic fibrosis is a constant pathological feature of chronic pancreatitis and the hyperactive stroma associated with pancreatic cancer. Strong evidence supports an important role of cyclooxygenase-2 (COX-2) and COX-2 generated prostaglandin E2 (PGE2) during pancreatic fibrosis. Pancreatic stellate cells (PSC) are the predominant source of extracellular matrix production (ECM), thus being the key players in both diseases. Given this background, the primary objective is to delineate the role of PGE2 on human pancreatic stellate cells (PSC) hyper activation associated with pancreatic cancer. This study showed that human PSC cells express COX-2 and synthesize high levels of PGE2. PGE2 stimulated PSC migration and invasion; expression of extra cellular matrix (ECM) genes and tissue degrading matrix metallo proteinases (MMP) genes. I further identified the PGE2 EP receptor responsible for mediating these effects on PSC. Using genetic and pharmacological approaches I identified the receptor required for PGE2 mediates PSC hyper activation. Treating PSC with Specific antagonists against EP1, EP2 and EP4, demonstrated that blocking EP4 receptor only, resulted in a complete reduction of PGE2 mediated PSC activation. Furthermore, siRNA mediated silencing of EP4, but not other EP receptors, blocked the effects of PGE2 on PSC fibrogenic activity. Further examination of the downstream pathway modulators revealed that PGE2 stimulation of PSC involved CREB and not AKT pathway. The regulation of PSC by PGE2 was further investigated at the molecular level, with a focus on COL1A1. Collagen I deposition by PSC is one of the most important events in pancreatic cancer. I found that PGE2 regulates PSC through activation of COL1A1 expression and transcriptional activity. Downstream of PGE2, silencing of EP4 receptor caused a complete reduction of COL1A1 expression and activity supporting the role of EP4 mediated stimulation of PSC. Taken together, this data indicate that PGE2 regulates PSC via EP4 and suggest that EP4 can be a better therapeutic target for pancreatic cancer to reduce the extensive stromal reaction, possibly in combination with chemotherapeutic drugs can further kill pancreatic cancer cells.

Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^