Regulation Of Prostaglandin Synthesis Research Articles

Cell type-specific endometrial transcriptome changes during initial recognition of pregnancy in the mare.

Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.

Novel insights into the mechanisms of pregnancy establishment: regulation of prostaglandin synthesis and signaling in the pig

Ovarian progesterone induces essential changes leading to a temporary state of uterine receptivity for conceptus implantation. Estrogens secreted by the porcine conceptus on days 11 and 12 of pregnancy provide the initial signal for maternal recognition of pregnancy and maintenance of a functional corpus luteum (CL) for continued production of progesterone. As prostaglandins F(2)(α) (PGF(2)(α)) and E(2) (PGE(2)) exert opposing actions on the CL, a tight control over their synthesis and secretion is critical either for the initiation of luteolysis or maintenance of pregnancy. One of the supportive mechanisms by which conceptus inhibits luteolysis is changing PG synthesis in favor of luteoprotective PGE(2). Conceptus PGE(2) could be amplified by PGE(2) feedback loop in the endometrium. In pigs, as in other species, implantation and establishment of pregnancy is associated with upregulation of expression of proinflammatory factors, which include cytokines, growth factors, and lipid mediators. The conceptus produces inflammatory mediators: interferon γ and interferon δ, interleukins IL1B and IL6, and PGs, which probably activate inflammatory pathways in the endometrium. The endometrium responds to these embryonic signals by enhancing further progesterone-induced uterine receptivity. Understanding the mechanisms of pregnancy establishment is required for translational research to increase reproductive efficiencies and fertility in humans and animals.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide–regulated prostaglandin secretion by cultured bovine oviductal epithelial cells

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)– or nitric oxide (NO)–regulated prostaglandin (PG), and nitrite/nitrate (NO 2/NO 3) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17β-estradiol (E 2; 10 −9 M) and/or progesterone (P 4; 10 −7 M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10 −5 M). Prostaglandin F 2α and PGE 2 secretion was measured in medium by ELISA. The pretreatment of cells with P 4 (progesterone), E 2 (17 β-estradiol), or E 2/P 4 augmented TNF-α–induced PGF 2α and PGE 2 secretion ( P < 0.01). The pretreatment of cells with E 2 or E 2/P 4 increased NONOate-induced PGF 2α and PGE 2 secretion ( P < 0.01). TNF-α induced NO 2/NO 3 production by BOECs. The pretreatment of cells with E 2 augmented only TNF-α–induced NO 2/NO 3 production ( P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10 −5 M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase ( P < 0.05) and endothelial and inducible NOSs ( P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS ( P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

Regulation of ovarian prostaglandin synthesis in the zebrafish: actions of 17α, 20β-dihydroxy-4-pregnen-3-one and insulin-like growth factor-1

Event Abstract Back to Event Regulation of ovarian prostaglandin synthesis in the zebrafish: actions of 17α, 20β-dihydroxy-4-pregnen-3-one and insulin-like growth factor-1 Nicholas C. Melnyk1, Andrea L. Lister1 and Glen J. Van Der Kraak1* 1 University of Guelph, Canada Oocyte maturation and ovulation are sequential events leading to the release of an oocyte that is ready to be fertilized. While earlier studies have shown that 17α, 20β-dihydroxy-4-pregnen-3-one (17,20β-P) and the insulin-like growth factor (IGF) system are regulators of oocyte maturation in the zebrafish (Danio rerio), it is not known whether these hormones play a role in the regulation of prostaglandin synthesis which is thought to mediate ovulation. The goal of this study was to determine in mid-vitellogenic (MV) and full grown (FG) zebrafish ovarian follicles whether 17,20β-P and human recombinant IGF-1 affect the expression of genes involved in prostaglandin biosynthesis including phospholipase A2 (cpla2), cycoloxygenase -1 (ptgs1) and cyclooxygenase 2 (ptgs2), or the levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). Both 17,20β-P and IGF-1 stimulated oocyte maturation in both size classes of follicles. In FG follicles, addition of 17,20β-P caused an increase in cpla2 expression, whereas IGF-1 increased ptgs2 expression and at high concentrations stimulated cpla2. There was an additive increase in cpla2 expression when both hormones were combined. Measurement of prostaglandin levels from the incubation medium revealed that both 17,20β-P and IGF-1 increased PGF2α production, and IGF-1 also induced PGE2 synthesis. Interestingly, 17,20β-P had no effect on cpla2 and ptgs2 expression and PGF2α production in MV follicles, whereas IGF-1 stimulated the expression of both cpla2 and ptgs2 and production of PGF2α in these follicles. Collectively, these results suggest that the regulation of prostaglandin biosynthesis in fish ovarian follicles may involve the coordinated actions of 17,20β-P and IGFs. Furthermore there are developmental differences in the responsiveness of MV and FG follicles to these regulators. Acknowledgements This work was supported by an NSERC Discovery Grant to Glen Van Der Kraak. Keywords: cyclooxygenase, Insulin-like growth factor, Maturation inducing steroid, Oocyte maturation, phospholipase, prostaglandin Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Gonadal development and gamete maturation Citation: Melnyk NC, Lister AL and Van Der Kraak GJ (2011). Regulation of ovarian prostaglandin synthesis in the zebrafish: actions of 17α, 20β-dihydroxy-4-pregnen-3-one and insulin-like growth factor-1. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00037 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Glen J Van Der Kraak, University of Guelph, Guelph, Canada, gvanderk@uoguelph.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nicholas C Melnyk Andrea L Lister Glen J Van Der Kraak Google Nicholas C Melnyk Andrea L Lister Glen J Van Der Kraak Google Scholar Nicholas C Melnyk Andrea L Lister Glen J Van Der Kraak PubMed Nicholas C Melnyk Andrea L Lister Glen J Van Der Kraak Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ceramide 1-Phosphate Is Required for the Translocation of Group IVA Cytosolic Phospholipase A2 and Prostaglandin Synthesis

Little is known about the regulation of eicosanoid synthesis proximal to the activation of cytosolic phospholipase A(2)alpha (cPLA(2)alpha), the initial rate-limiting step. The current view is that cPLA(2)alpha associates with intracellular/phosphatidylcholine-rich membranes strictly via hydrophobic interactions in response to an increase of intracellular calcium. In opposition to this accepted mechanism of two decades, ceramide 1-phosphate (C1P) has been shown to increase the membrane association of cPLA(2)alpha in vitro via a novel site in the cationic beta-groove of the C2 domain (Stahelin, R. V., Subramanian, P., Vora, M., Cho, W., and Chalfant, C. E. (2007) J. Biol. Chem. 282, 20467-204741). In this study we demonstrate that C1P is a proximal and required bioactive lipid for the translocation of cPLA(2)alpha to intracellular membranes in response to inflammatory agonists (e.g. calcium ionophore and ATP). Last, the absolute requirement of the C1P/cPLA(2)alpha interaction was demonstrated for the production of eicosanoids using murine embryonic fibroblasts (cPLA(2)alpha(-/-)) coupled to "rescue" studies. Therefore, this study provides a paradigm shift in how cPLA(2)alpha is activated during inflammation.

Differential Regulation of Prostaglandin Production Mediated by Corticotropin-Releasing Hormone Receptor Type 1 and Type 2 in Cultured Human Placental Trophoblasts

Prostaglandin (PG) production by intrauterine tissues plays a key part in the control of pregnancy and parturition. The present study was to investigate the role of placenta-derived CRH and CRH-related peptides in the regulation of PG synthesis and metabolism. We found that placental trophoblasts expressed both CRH-R1 and CRH-R2. Treatment of cultured placental cells with either a CRH or urocortin I (UCNI) antibody resulted in a significant decrease in PGE2 release. Both CRH and UCNI antibodies significantly decreased mRNA and protein expression of synthetic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2 and increased mRNA and protein expression of 15-hydroxyprostaglandin dehydrogenase (PGDH), the key enzyme of PG metabolism. CRH-R1/-R2 antagonist astressin and CRH-R1 antagonist antalarmin significantly inhibited PGE2 release, whereas CRH-R2 antagonist astressin-2b had no effect on PGE(2) release. Administration of astressin decreased expression of cPLA2 but had no effect on COX-2 expression. Antalarmin reduced cPLA2 and COX-2 expression, whereas astressin-2b did not alter cPLA2 expression but increased COX-2 expression. PGDH expression was enhanced by these three antagonists. Cells treated with exogenous CRH and UCNI showed an increase in PGE(2) release and expression of cPLA2 and COX-2 but a decrease in PGDH expression. UCNII and UCNIII had no effect on PGE2 release but decreased COX-2 and PGDH expression. Our results suggested CRH and CRH-related peptides act on CRH-R1 and CRH-R2 to exert different effects on PG biosynthetic enzymes cPLA2 and COX-2 and thereby modulate output of PGs from placenta, which would be important for controlling pregnancy and parturition.

Stress Mediators Regulate Brain Prostaglandin Synthesis and Peroxisome Proliferator-Activated Receptor-γ Activation after Stress in Rats

Stress exposure leads to oxidative/nitrosative and neuroinflammatory changes that have been shown to be regulated by antiinflammatory pathways in the brain. In particular, acute restraint stress is followed by cyclooxygenase (COX)-2 up-regulation and subsequent proinflammatory prostaglandin (PG) E2 release in rat brain cortex. Concomitantly, the synthesis of the antiinflammatory prostaglandin 15d-PGJ(2) and the activation of its nuclear target the peroxisome proliferator-activated receptor (PPAR)-gamma are also produced. This study aimed to determine the possible role of the main stress mediators: catecholamines, glucocorticoids, and excitatory amino acids (glutamate) in the above-mentioned stress-related effects. By using specific pharmacological tools, our results show that the main mediators of the stress response are implicated in the regulation of prostaglandin synthesis and PPARgamma activation in rat brain cortex described after acute restraint stress exposure. Pharmacological inhibition (predominantly through beta-adrenergic receptor) of the stress-released catecholamines in the central nervous system regulates 15d-PGJ(2) and PGE(2) synthesis, by reducing COX-2 overexpression, and reduces PPARgamma activation. Stress-produced glucocorticoids carry out their effects on prostaglandin synthesis through their interaction with mineralocorticoid and glucocorticoid receptors to a very similar degree. However, in the case of PPARgamma regulation, only the actions through the glucocorticoid receptor seem to be relevant. Finally, the selective blockade of the N-methyl-d-aspartate type of glutamate receptor after stress also negatively regulates 15d-PGJ(2) and PGE(2) production by COX-2 down-regulation and decrease in PPARgamma transcriptional activity and expression. In conclusion, we show here that the main stress mediators, catecholamines, GCs, and glutamate, concomitantly regulate the activation of proinflammatory and antiinflammatory pathways in a possible coregulatory mechanism of the inflammatory process induced in rat brain cortex by acute restraint stress exposure.

Regulation of prostaglandin synthesis by interleukin-1α in bovine endometrium during the estrous cycle

Interleukin (IL)-1 has been suggested to participate in regulation of many reproductive functions. To investigate the possible role of IL-1α as a local regulator in bovine endometrium, we determined the effects of IL-1α on prostaglandin (PG) E2 and PGF 2α output by the bovine endometrium at different stages of the estrous cycle. The expressions of IL-1α and IL-1 receptor type 1 (IL-1RT1) mRNA in bovine endometrium were also studied. Bovine uteri were classified into six stages (estrus: day 0; early luteal: days 2–3; developing luteal: days 5–6; mid luteal: days 8–12; late luteal: days 15–17; and follicular: days 19–21). After 1 h of pre-incubation, endometrial tissues (20–30 mg) were exposed to 0 or 10 ng/ml IL-1α for 4 h. IL-1α significantly stimulated PGE2 output throughout the luteal stages, with the highest response during the mid luteal stage, while it did not stimulate PGE2 output during the estrus and the follicular stage. On the other hand, IL-1α significantly enhanced PGF 2α output throughout the estrous cycle except in the endometrium from the mid luteal stage, with the highest response at the follicular stage ( P < 0.001). The treatment of endometrial tissue with IL-1α resulted in an increase of the PGE2:PGF 2α ratio at the mid luteal stage, and in a decrease during the late luteal and follicular stages of the estrous cycle. A semiquantitative reverse transcription-polymerase chain reaction revealed that IL-1α and IL-1RT1 mRNA are expressed in the endometrium throughout the estrous cycle. IL-1α mRNA expression was greater in the early luteal stage than in the estrus, late luteal, and follicular stages ( P < 0.05). IL-1RT1 mRNA was greater in the late luteal stage than in the other stages ( P < 0.05). The overall results suggest that IL-1α is produced in bovine endometrium throughout the estrous cycle, and plays some roles not only in maintenance of CL, but also in luteolysis by regulating the local PGE2:PGF 2α ratio in bovine endometrium during the estrous cycle.

Deficient prostaglandin E2 production by bronchial fibroblasts of asthmatic patients, with special reference to aspirin-induced asthma

Background: Regulation of prostaglandin synthesis and the activation of human airway fibroblasts associated with the remodeling of the bronchi play an important role in asthma. Objective: We sought to assess the cyclooxygenase pathways in airway fibroblasts of patients with bronchial asthma. Methods: Generation of prostaglandin E2 (PGE2) and pros-taglandin D2 (PGD2) by bronchial fibroblasts was measured by means of mass spectrometry in culture supernatants, and cyclooxgenases expression was estimated by means of RT-PCR and immunoblotting. The cells were isolated from 3 groups of subjects: nonasthmatic patients (n = 10), patients with aspirin-tolerant asthma (ATA, n = 9), and patients with aspirin-intolerant asthma (AIA, n = 7). Results: The cytomix (LPS, 5 □g/mL; IL-1□, 5 ng/mL; and TNF-□, 10 ng/mL; 18 hours) stimulated the production of prostaglandins. Asthmatic patients were characterized by low capacity to produce PGE2 after cytomix stimulation. In the nonasthmatic patient group the mean PGE2 production was 32 ± 33 ng/mL (35-fold of the basic production), in the ATA group it was 16 ± 18 ng/mL (16-fold), and in the AIA group it was only 5.3 ± 3.6 ng/mL (4-fold). The mean concentration of PGD2 for nonasthmatic patients, patients with ATA, and patients with AIA was 0.18 ± 0.16 ng/mL (4.7-fold of the basic production), 0.18 ± 0.14 ng/mL (4.2-fold), and 0.235 ± 0.19 ng/mL (1.9-fold), respectively. The observed difference was not due to insufficient cyclooxygenase 2 expression because all groups had similar levels of its mRNA and protein. The patients with AIA had low expression levels of cyclooxygenase 1 protein but not of its mRNA. The PGE2/PGD2 concentration ratio increased after cytomix stimulation in all groups but was significantly less in patients with AIA than in patients with ATA. Conclusions: Our results point to a deficient PGE2 production under proinflammatory conditions in asthmatic airways. This could weaken local defensive mechanisms and promote cysteinyl leukotriene overproduction. (J Allergy Clin Immunol 2003;111:1041-8.)

Regulation of prostaglandin synthesis in the human uterus.

Prostaglandins are important regulators of many aspects of reproductive processes from ovulation, fertilization and pregnancy recognition to labor and parturition. These biologically potent compounds are members of the large family of eicosanoids, derived from polyunsaturated fatty acids, principally arachidonic acid, found in the membrane phospholipids of virtually every cell of the human body, accounting for the ubiquity of prostaglandins, which act in a paracrine or autocrine fashion via discrete receptors. The availability of specific prostaglandins in various cells and tissues depends on the presence and activity of specific enzymes that convert a common precursor to the end product, as well as on the rate of enzymatic or spontaneous inactivation of the bioactive compounds. Here we offer a brief review of the regulation of prostaglandin generation in human uterine tissues, focusing on their role in labor and parturition at term and preterm.

Regulation of prostaglandin synthesis in the human uterus

Regulation of prostaglandin synthesis in the human uterus

Regulation of prostaglandin synthesis and cell adhesion by a tryptophan catabolizing enzyme

BackgroundThe tryptophan catabolizing enzyme, indoleamine 2,3, dioxygenase (IDO) is one of two mammalian enzymes, which can catabolize the rarest essential amino acid, tryptophan. IDO is inducible by cytokines such as interferon-γ and plays a role in inflammation and maternal tolerance of fetal allografts, although its exact mode of action is unclear. Therefore, we investigated the circumstances under which IDO is expressed in vitro together with the effects of overexpression of IDO on the growth and morphology of cells.ResultsOverexpression of IDO in the murine macrophage cell line RAW 264.7 and the murine fibrosarcoma cell line MC57, resulted in the growth of macroscopic cell foci, with altered cell adhesion properties. The expression of IDO was also detected during adhesion of wild type, nontransfected cells in tissue culture to standard cell growth substrates. Inhibition of this expression, likewise resulted in alterations in cell adhesion. Overexpression of IDO or inhibition of endogenous IDO expression was accompanied by changes in metalloproteinase expression and also in the expression and activity of the cyclooxygenase enzymes. In the case of RAW cells, IDO effects on cell growth could be reversed by adding back prostaglandins.ConclusionsThese results suggest that catabolism of the rarest essential amino acid may regulate processes such as cell adhesion and prostaglandin synthesis.

THE ROLE OF NITRIC OXIDE IN ARTICULAR CARTILAGE DAMAGE

The production of large amounts of NO in vitro by cytokine-activated chondrocytes has been established. In vitro studies suggest that NO compromises chondrocyte survival. The role of NO in regulating matrix biosynthesis and degradation has received much attention. Most studies indicate that NO is at least partly responsible for IL-1-induced suppression of glycosaminoglycan and collagen synthesis. NO also may be involved as a mediator of IL-1-induced expression of MMP, mRNA, and protein and may contribute as an activator of the latent forms of the enzymes. Although the interaction of NO and prostaglandins is of considerable interest, current data are inconclusive with respect to the role of NO in the regulation of prostaglandin synthesis, although it seems clear that prostaglandin is not involved in NO synthesis. It is important to note that NO does have protective effects in cartilage and other tissues. Under certain conditions, NO may have anabolic and anticatabolic effects in cartilage. In other tissues, notably in skin and muscle, NO has been found to have a stimulatory role in extracellular matrix repair. In antimicrobial defense, in general, and in bacterial arthritis specifically, NO is an important protective molecule. Production of NO in arthritis-affected cartilage and synovium is a consistent feature of human and experimentally induced arthritis. The production of NO is associated with matrix degradation and chondrocyte apoptosis. The administration of NO synthase inhibitors in experimentally induced arthritis has resulted in reduction of synovial inflammation and destruction of cartilage and bone.

Transforming growth factor beta-1 and -2 regulation of prostaglandin synthesis in cultured bovine endometrial cells and cytokine expression in lymphocytes

Transforming growth factor beta-1 and -2 regulation of prostaglandin synthesis in cultured bovine endometrial cells and cytokine expression in lymphocytes

Regulation of prostaglandin production in intact fetal membranes by interleukin-1 and its receptor antagonist.

There is strong evidence for the involvement of inflammatory mediators such as interleukin (IL)-1 in the biochemical mechanisms of parturition. Therefore the effects of the IL-1 family (IL-1alpha (1 ng/ml), IL-1beta (1 ng/ml) and the IL-1 receptor antagonist (IL-1ra) (10 ng/ml)) on the regulation of prostaglandin synthesis in term human fetal membranes were investigated. It was found that, after 4 h of culture, IL-1beta increased prostaglandin E2 (PGE2) output approximately twofold. This was associated with both a significant increase in cyclo-oxygenase-2 (COX-2) mRNA levels (approximately fourfold compared with control) and translocation of cytoplasmic phospholipase A2 (cPLA2) from the cytosol to the membrane fraction. IL-1alpha was less effective than IL-1beta at stimulating PGE2 production through similar mechanisms. IL-1ra had no effect on PGE2 output. However, in combination treatments, IL-1ra did not inhibit IL-1alpha- or IL-1beta-stimulated PGE2 output, and increased PGE2 production further compared with IL-1beta alone. IL-1ra decreased IL-1beta-induced COX-2 mRNA expression by about half and significantly increased cPLA2 protein levels, as detected by immunoblotting, when used alone and together with IL-1beta. These results suggest that IL-1ra has partial agonist properties when used together with IL-1alpha and IL-1beta in fetal membranes by increasing cPLA2 protein levels, which leads to an increase in the production of prostaglandins.

Modulation of endothelial prostaglandin synthesis by corticotropin releasing factor and antagonists

Corticotropin releasing factor (CRF) is a hypothalamic hormone that also displays autocrine/paracrine roles at peripheral sites. High concentrations of CRF have been identified in endothelial cells and other inflammatory tissues. We investigated the effects of CRF and antagonists in the regulation of prostaglandin synthesis in bovine aortic endothelial cells, and also characterized the binding of CRF in these cells. Interleukin-1α increased prostacyclin (prostaglandin I 2) synthesis in endothelial cells and this response to interleukin-1α was abolished by simultaneous exposure to CRF. The effect of CRF on interleukin-1α-induced prostaglandin synthesis was antagonised by the CRF receptor antagonist α-helical CRF-(9–41). In addition, this as well as another CRF receptor antagonist, namely [ d-Phe 12]CRF-(12–41), when applied alone at low concentrations inhibited the interleukin-1α-induced prostaglandin synthesis similarly to CRF, suggesting partial agonistic action. Binding of [ 125 I ]-labeled CRF in endothelial cells was saturable and fitted a two sites model. K d for the higher-affinity class of receptors was 0.2±0.02 nM, and B max 0.79±0.095 fmol/mg protein. The lower-affinity class of receptors had a K d of 1.77±0.14 μM and B max 0.97±0.12 fmol/mg protein. These findings suggest a direct role for CRF in the local regulation of inflammation.

Synthesis of an unsaturated fatty acid analogue (18-(4′-azido-2′-hydroxybenzoylamino)-oleic acid) and its interaction with lysophosphatidylcholine: acyl-CoA-O-acyltransferase

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional Cg-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue. The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.

Increased renal and vascular cytosolic phospholipase A2 activity in rats with cirrhosis and ascites.

Indirect evidence suggests that the renal and vascular production of prostaglandins is increased in cirrhosis with ascites. However, the activity of the enzymes regulating the prostaglandin pathway has not been investigated in cirrhosis. The aim of the current study was to determine the activity of phospholipase A2 (PLA2), the key enzyme in the regulation of prostaglandin synthesis, in kidney and vascular tissue obtained from rats with carbon tetrachloride-induced cirrhosis and ascites (n = 9) and control rats (n = 6). PLA2 activity was assayed in vitro using [14C]arachidonyl-phosphatidylcholine (PC) and [14C]arachidonyl-phosphatidylethanolamine (PE) as substrates in the presence of Ca2+. Kidneys from cirrhotic rats had significantly higher PLA2 activity compared with control rats, with both PC and PE (35 +/- 5 and 40 +/- 6 vs. 21 +/- 2 and 26 +/- 3 pmol/mg/min, respectively; P < .05 for both). PLA2 activity was increased in the renal cortex as well as in the renal medulla. Fractionation of the kidney extracts by Mono-Q anion-exchange chromatography showed that the elution position of PLA2 activity corresponded to the cytosolic PLA2 isoform (cPLA2). Increased amounts of cPLA2 protein were found in kidney extracts immunoblotted with an anti-cPLA2 antibody However, reverse-transcriptase polymerase chain reaction (RT-PCR) analysis did not detect any difference in cPLA2 mRNA. PLA2 activity was also higher in aortic tissue from cirrhotic rats than in controls (PC 38 +/- 5 vs. 26 +/- 1 and PE 66 +/- 8 vs. 41 +/- 3 pmol/mg/min; P < .05 for both). Incubation of renal and aortic extracts from cirrhotic rats with anti-cPLA2 antibody reduced PLA2 activity by 64% and 88%, respectively. In conclusion, PLA2 activity is increased in kidneys and vascular tissue from cirrhotic rats with ascites. This can be accounted for by an induction of cPLA2, which would mediate, at least in part, the increased renal and vascular production of prostaglandins in cirrhosis.

Role and regulation of prostaglandin synthesis in the mitogenic response of ovarian granulosa cells to transforming growth factor alpha.

Prostaglandins (PGs) are important regulators in a variety of ovarian functions including steroidogenesis, ovulation and luteolysis (1–4). PG synthesis and secretion are under the control of gonadotropins, and transforming growth factors(3,5). Although considerable attention has been focused on the role of PGs in the regulation of follicular rupture in the mammalian ovary (3), whether their secretion and action are involved in the activation of granulosa cell proliferation and accompanying follicular wall remodelling processes are uncertain. We have previously shown that TGFα and TGFβ play important roles in the regulation of granulosa cell proliferation and plasminogen activator (PA) activity during ovarian follicular development (6,7). Whether PGs are involved in these regulations needs to be clarified.

Preferential esterification of arachidonic acid into ethanolamine phospholipids in epithelial cells from ovine endometrium.

In sheep, the pulsatile release of prostaglandin F2 alpha by the endometrium is necessary to achieve luteolysis which occurs at the end of the oestrous cycle. The production of prostaglandins is known to depend upon the availability of arachidonic acid, the fatty acid precursor of prostaglandin biosynthesis. Consequently, the mechanisms controlling intracellular amounts of arachidonate may be involved in the regulation of prostaglandin synthesis. Since arachidonic acid is mostly found in phospholipids and the endometrial epithelium is the primary source of prostaglandin F2 alpha during luteolysis, the fate of arachidonic acid when incorporated into epithelial cells from the ovine uterus was investigated. Endometrial epithelial cells isolated from cyclic ewes at day 15 after oestrus were cultured in the presence of [3H]arachidonic acid. Incorporation and distribution of the radiolabelled arachidonic acid into the various phospholipid classes were examined using HPLC. We observed that ethanolamine glycerophospholipids contained 61% of the total tritiated arachidonic acid incorporated into cellular lipids, whereas phosphatidylinositols, phosphatidylcholines and phosphatidylserines contained 17%, 13% and 4.7%, respectively. In addition, the radioactivity measured within phosphatidylethanolamines was preferentially detected in the 1-alkenyl-2-acyl (44%) forms of ethanolamine phospholipids, also called plasmalogens. The kinetic study of arachidonic acid uptake into ethanolamine phospholipids showed that arachidonic acid was rapidly esterified into the diacyl forms and then uptake decreased, whereas the incorporation increased continuously into the plasmalogen forms for at least 24 h. These results demonstrate that the primary pool of esterified arachidonic acid is found in ethanolamine plasmalogens of epithelial cells from the ovine endometrium. The high arachidonate content of ethanolamine plasmalogens suggests that these phospholipids play a crucial role in the control of arachidonic acid availability and ultimately in the regulation of prostaglandin synthesis.