Primary Prostaglandins Research Articles

The five primary prostaglandins stimulate contractions and phasic activity of the urinary bladder urothelium, lamina propria and detrusor

BackgroundInflammation is often associated with several bladder dysfunctions, including overactive bladder (OAB) and interstitial cystitis/bladder pain syndrome (IC/PBS). As such, inflammation of the bladder and the actions of inflammatory mediators may contribute to the development of urinary symptoms. This study assessed the actions of PGE2, PGF2, PGD2, TXA2, and PGI2 on urinary bladder urothelium with lamina propria (U&LP), and detrusor smooth muscle.MethodsStudies were carried out using isolated tissue baths, where strips of porcine bladder U&LP or detrusor were exposed to varying concentrations of prostaglandin agonists (1 μM and 10 μM).ResultsAll assessed prostaglandin agonists contracted both the U&LP and detrusor smooth muscle, with the rank order of contractile response effectiveness as: PGE2 > PGF2α > TXA2 > PGD2 > PGI2. In U&LP, treatment with PGE2 (10 μM) increased tonic contractions by 1.36 ± 0.09 g (n = 42, p < 0.001) and phasic contractions by 40.4 ± 9.6% (n = 42, p < 0.001). In response to PGF2α (10 μM), U&LP tonic contractions increased by 0.79 ± 0.06 g (n = 14, p < 0.001) and phasic activity by 13.3% ± 5.3% (n = 15, p < 0.05). In detrusor preparations, PGE2 (10 μM) increased tonic contractions by 1.32 ± 0.13 g (n = 38, p < 0.001) and PGF2α (10 μM) by 0.97 ± 0.14 g (n = 12, p < 0.001). Only 34% (n = 48) of all detrusor preparations exhibited spontaneous activity prior to the addition of any agonist at a frequency of 2.03 ± 0.12 cpm. In preparations that did not exhibit initial phasic activity, all of the prostaglandin agonists were capable of commencing phasic activity.ConclusionsThe urinary bladder U&LP and detrusor respond to a variety of prostaglandin agonists, with their activation resulting in direct contractions, as well as increases to spontaneous contractile activity. This study presents the prostaglandin receptor system as a potential therapeutic target for lower urinary tract dysfunction.

Prostaglandins in asthma and allergic diseases

Prostaglandins are synthesized through the metabolism of arachidonic acid via the cyclooxygenase pathway. There are five primary prostaglandins, PGD2, PGE2, PGF2, PGI2, and thromboxane B2, that all signal through distinct seven transmembrane, G-protein coupled receptors. The receptors through which the prostaglandins signal determines their immunologic or physiologic effects. For instance, the same prostaglandin may have opposing properties, dependent upon the signaling pathways activated. In this article, we will detail how inhibition of cyclooxygenase metabolism and regulation of prostaglandin signaling regulates allergic airway inflammation and asthma physiology. Possible prostaglandin therapeutic targets for allergic lung inflammation and asthma will also be reviewed, as informed by human studies, basic science, and animal models.

Astrocytes synthesize primary and cyclopentenone prostaglandins that are negative regulators of their proliferation

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ± 36 pg/mg PGE2, 344 ± 24 PGD2) and cyclopentenone PGs (cyPGs) (87 ± 17 15d-PGJ2, 308 ± 23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 μM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.

Peroxide Tone in Human Inferior Nasal Turbinate with Allergy

Peroxide Tone in Human Inferior Nasal Turbinate with Allergy Background: The nose is chronically exposed to oxidative stress, which can easily lead to reactive oxygen species (ROS)-mediated damage and lipid oxidative damage of the upper airway. ROS may also participate in various diseases, including those of the airway, although many details are not yet known. ROS is generated by various enzymatic reactions and chemical processes. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) are representative scavengers. ROS are also generated through arachidonic acid cascades. One of the primary prostaglandins (PG), PGD2, is the major PG in most types of tissue, including the nose. We attempted to evaluate the peroxide tone by measuring these factors. Methods: A total of 42 Japanese patients with and without nasal allergies were enrolled in this study. We determined the contents of lipid peroxide (LPO) and PGD2, as well as the activities of SOD, catalase, and GSH-Px of the anterior portion of the mucosa of the inferior turbinate, obtained by inferior turbinotomy. Results: LPO and PGD2 contents increased significantly in the nasal allergy group. No statistically significant difference appeared in the activities of SOD, catalase, and GSH-Px were demonstrated in the nasal allergy group compared with the subjects without nasal allergy. Conclusion: The imbalance of the peroxide tone in the nasal mucosa caused by stimulation of the cyclooxygenase pathway of the arachidonic acid cascade and ROS formation was demonstrated. Moreover, the increased number of ROS was not well metabolized in the nasal mucosa with allergy.

Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2 synthase activity of aldo-ketoreductase 1B1

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

Profile of eicosanoid generation in aspirin-intolerant asthma and anaphylaxis assessed by new biomarkers

It has recently demonstrated that a free radical-mediated pathway generates prostaglandins (PGs) and the corresponding prostaglandin enantiomers (ent-PGs). Aspirin-intolerant asthma and anaphylaxis accompany PGD(2) overproduction, possibly associated with mast cell activation via the COX pathway. However, free radical-mediated PG generation in the pathophysiology of these diseases, which can be demonstrated by measuring urinary ent-PGF(2)alpha, has not been reported. To evaluate the characteristic profile of eicosanoid generation via the COX and/or free radical-mediated pathway underlying aspirin-intolerant asthma and anaphylaxis. A comparative group analysis consisted of asthma (n = 17) and anaphylaxis (n = 8, none with aspirin-induced anaphylaxis) cases. Urinary eicosanoid concentrations were quantified as follows: 2,3-dinor-9alpha,11beta-PGF(2) by gas chromatography-mass spectrometry; leukotriene E(4), 9alpha,11beta-PGF(2), and PGs by enzyme immunoassay. 2,3-Dinor-9alpha,11beta-PGF(2) is a more predominant PGD(2) metabolite in urine than 9alpha,11beta-PGF(2). At baseline, the aspirin-intolerant asthma group (n = 10) had significantly higher leukotriene E(4) and lower PGE(2) concentrations in urine than the aspirin-tolerant asthma group. During the reaction, the urinary concentrations of leukotriene E(4) and PGD(2) metabolites correlatively increased, but with markedly different patterns of the mediator release, in the aspirin-intolerant asthma group and the anaphylaxis group, respectively. The urinary PGD(2) metabolites and primary PGs were significantly decreased in the aspirin-tolerant asthma group. Urinary ent-PGF(2)alpha concentrations were significantly increased in the anaphylaxis group but not the aspirin-intolerant asthma group. When assessed by urinary 2,3-dinor-9alpha,11beta-PGF(2), PGD(2) overproduction during aspirin-intolerant bronchoconstriction was clearly identified, regardless of COX inhibition. It is evident that free radical-mediated PG generation is involved in the pathophysiology of anaphylaxis.

Cyclooxygenase-2-Derived Prostaglandin F 2α

See related article, pages 228–235 Soluble vasoactive factors derived from vascular endothelium modulate the tone of underlying smooth muscle cells. Stimulation of intact, ie, endothelialized, vessels with acetylcholine causes vasodilation by release of endothelium-derived relaxing factors (EDRFs), mostly NO and hyperpolarizing factor(s) (EDHF). The endothelium also produces contractile factors (EDCF) of different origin.1 Thus, any reduced endothelium-dependent relaxation or even vasoconstriction could result from either loss of EDRFs, (enhanced) generation of EDCFs, or a combination of both. EDCF-mediated vasoconstriction might prevail with increasing age, as well as disturbed endothelial function, for example, in hypertension1,2 or type 2 diabetes.3 In addition to reduced generation of vasodilator prostaglandins (PGs), such as PGI2, enhanced production of vasoconstrictor prostanoids has been described.4,5 Oxygen-derived free radicals and isoprostanes, generated by exaggerated oxidative stress, are further members of the EDCF family. Thus, EDCFs represent an inhomogeneous group of compounds with different chemical identity. In the few last years, vasocontractile prostanoids, generated by endothelial cyclooxygenase(s) (COX[s]), have come into focus as significant EDCFs.1 One reason for this is the upregulation of endothelial COXs and enzymes of PG-endoperoxide catabolism during the aging process and hypertension with enhanced generation of these compounds6,7; another reason is the low selectivity of prostanoids for their membrane receptors. Vasoconstrictor prostanoids act on vascular smooth muscle cells via the thromboxane-prostanoid (TP) receptor.8 However, reactive oxygen species and other primary PGs, including PGI2 at higher concentrations,9 may also activate this receptor and cause vasoconstriction. In this issue of Circulation Research , Wong and colleagues10 demonstrate generation of a …

Optimization of a solid phase extraction procedure for prostaglandin E 2, F 2α and their tissue metabolites

The primary prostaglandins PGE 2 and PGF 2α are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE 2, PGF 2α and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol–water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to ≥90%. This SPE method is the first that has been optimized by systematic elution studies for PGE 2, PGF 2α and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE 2 and PGF 2α from complex biological matrices.

Misidentification of prostamides as prostaglandins

Prostaglandins and endogenous cannabinoid metabolites share the same lipid backbone with differing polar head groups at exactly the position through which a large molecule is attached to provide antigenicity and thus raise antisera. Hence, we hypothesized that antisera raised against prostaglandins linked to a large molecule such as BSA at the carboxyl functional group would also recognize endogenous cannabinoid metabolites and lead to highly misleading interpretations of data. We found major cross-reactivity of commercial antisera raised to prostaglandins with endocannabinoid metabolites. Furthermore, in a well-characterized cell line (WISH) or primary amnion tissue explants, endocannabinoid treatment led to increased production of endocannabinoid metabolites as opposed to primary prostaglandins. This was apparent only after separation of products by thin-layer chromatography, because they measured as prostaglandins by radioimmunoassay. These findings have major implications for our interpretation of data in situations in which these prostaglandin-like molecules are formed, and they stress the need for chromatographic or spectrometric confirmation of prostaglandin production detected by antibody-based methods.

Increase in 15-hydroxyprostaglandin dehydrogenase activity in the ovine placentome at parturition and effect of oestrogen

Type 1 NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is the key enzyme for metabolism of active primary prostaglandins to inactive forms in gestational tissues. The present study examined the activity and immunolocalization of PGDH in the ovine placenta, fetal membranes and uterus over the latter half of pregnancy, and its potential regulation by oestradiol. Placenta, fetal membranes and myometrium were collected from sheep with known single insemination dates on days 70, 100 and 135 of gestation and in active labour demonstrated by electromyographic activity. In addition, chronically catheterized fetuses were infused with oestradiol (100 microgram kg(-1) per 24 h) (n = 5) or saline vehicle into the fetus from day 120 to day 125. PGDH activity measured in placental extracts remained constant from day 70 to day 135 of gestation, and then significantly (P < 0.05) increased by 300% in active labour. Immunoreactive PGDH was localized in the placentome at all stages and was present predominantly in the fetal component of the placentome in uninucleate, but not in binucleate, trophoblast cells. Similarly, in the fetal membranes PGDH immuno-reactivity was present in the uninucleate trophoblast but not in the binucleate cells of the chorion. PGDH immunostaining was also present in the endometrial luminal epithelium, in the smooth muscle of the myometrium, and the glandular epithelium of the cervix. Infusion of oestradiol into the fetal circulation from day 120 to day 125 of gestation had no effect on placental PGDH activity. Immunohistochemistry was used to localize oestrogen receptor alpha in intrauterine tissues to investigate further the failure of oestradiol to increase PGDH activity. Immunoreactive oestrogen receptor alpha was not present in the fetal component of the placenta, although it was expressed in adjacent maternal-derived cells. It is concluded that (1) PGDH activity increases in late gestation; (2) PGDH is expressed in uninucleate trophoblast cells in the ovine placenta and fetal membranes, and also in the maternal endometrial epithelium and stroma, myometrium and cervix; (3) oestrogen receptor alpha is not expressed in fetal cells in the placenta or fetal membranes; and (4) the increase in PGDH activity is not regulated by oestradiol administered to the fetus.

Transfer and Metabolism of Prostaglandin E2in the Dual Perfused Human Placenta

Prostaglandins (PGs) are potent paracrine hormones that are important for the control of several functions in the uterus and fetus during pregnancy and parturition. PGs are rapidly metabolized to inactive metabolites by prostaglandin dehydrogenase (PGDH). However, the regulation of transfer and metabolism of PGs across the placenta is not well understood. This study used an in vitro dual perfused human placental cotyledon preparation to examine the production of the potent vasoactive and myometrial stimulants PGE2and PGF2α, transfer of PGs from the maternal to the fetal circulation and the metabolism of PGs by PGDH. Secretion of PGE2was greater into the fetal compared to the maternal circulation. PGE2output was higher than PGF2αand concentrations of PGE2and PGF2αmetabolites (PGEM and PGFM) were greater in both fetal and maternal outputs when compared to the primary prostaglandins. Infusion of PGE2into the maternal circulation did not result in increased PGE2efflux but PGEM was output was increased, demonstrating a rapid and efficient metabolism by the placenta. There was no significant transfer of PGE2across to the fetal circulation, although there was some transfer but in the form of inactivated PGEM. There was no significant interconversion of PGE2to PGF2αby the 9-keto-reductase pathway. Expression of PGDH as detected by immunoblot was high in placenta. This PGDH was localized throughout the syncytiotrophoblast at the fetal–maternal interface and also in extravillous trophoblast cells. The presence of PGDH at this site acts to stabilize output of primary PG from the placenta and also as a barrier preventing transfer to the fetal circulation, resulting in the separation of PG homeostasis in the fetus and mother.

Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta.

We have demonstrated previously that cyclo-oxygenase-2 (COX2), the rate-limiting enzyme in the biosynthesis of prostaglandins (PGs), is essential for blastocyst implantation and decidualization. However, the candidate PG(s) that participates in these processes and the mechanism of its action remain undefined. Using COX2-deficient mice and multiple approaches, we demonstrate herein that COX2-derived prostacyclin (PGI2) is the primary PG that is essential for implantation and decidualization. Several lines of evidence suggest that the effects of PGI2 are mediated by its activation of the nuclear hormone receptor PPARdelta, demonstrating the first reported biologic function of this receptor signaling pathway.

ChemInform Abstract: Synthesis and Pharmacological Activities of 13‐Dehydro Derivatives of Primary Prostaglandins.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and pharmacological activities of 13-dehydro derivatives of primary prostaglandins.

13-Dehydro derivatives of prostaglandin E1, E2, E3, F1 alpha and F2 alpha were synthesized. Compared with natural prostaglandins, 13-dehydro analogues were found to exhibit more potent inhibitory activity against human platelet aggregation and relaxation of guinea-pig isolated trachea, while they showed less potent activity of contraction of guinea-pig isolated ileum.

IFN- τ increases PGE 2 production and COX-2 gene expression in the bovine endometrium in vitro

Prostaglandins (PGs) are well known for their role in reproductive processes. At the time of pregnancy recognition, PGF 2 α is luteolytic and PGE 2 may be antiluteolytic and luteotropic. During the preimplantation period, interferon-tau (IFN- τ) is produced by the conceptus and plays a crucial role in maternal recognition of pregnancy in domestic ruminants. We have demonstrated previously that recombinant bovine and ovine interferon-tau (rbIFN- τ and roIFN- τ) stimulate PGE 2 production in epithelial cells, changing the primary PG produced by these cells from F 2 α to E 2. In stromal cells, where PGE 2 is the major PG produced, roIFN- τ induced an increase of both types of PGs. The aim of this paper is to identify the possible involvement of cyclooxygenases (COXs) in the modulation of PG production by trophoblastic interferons. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses (1, 10 and 20 μg/ml) of roIFN- τ. PG levels in the culture media were measured by enzyme immunoassays (EIA) and total RNA was extracted from the cells. Northern blot analysis was performed to quantify cyclooxygenase COX-1 (constitutive), COX-2 (inducible) and phospholipase A 2 (PLA 2) messenger RNA (mRNA) production in response to treatment. The results indicate that roIFN- τ treatment did not affect COX-1 and PLA 2 mRNA production in either cell type, whereas COX-2 expression was upregulated in both. The up-regulation of COX-2 transcript was greater in stromal than in epithelial cells. The increase in COX-2 mRNA levels was concurrent with increased production of PGE 2 and PGF 2 α in stromal cells and principally PGE 2 in epithelial cells. Furthermore, addition of indomethacin (1 μM) and a specific COX-2 inhibitor (NS-398, 1 μM) blocked the roIFN- τ-stimulation of PG production in both cell types. The mechanism whereby elevated COX-2 expression results in a selective increase of PGE 2 in epithelial cells remains to be elucidated. In stromal cells, an increase in COX-2 mRNA levels may explain increased PG production. The overall effect of roIFN- τ in the two cell types is a net increase in PGE 2 output.

Chorionic prostaglandin catabolism is decreased in the lower uterine segment with term labour

We have examined the hypothesis that regional differences in 15-hydroxyprostaglandin dehydrogenase (PGDH) activities occur within the human fetal membranes. Further, we reasoned that a specific reduction in PGDH in the fetal membranes at the lower uterine segment might occur with labour, providing a potential mechanism for local generation of primary prostaglandins (PG) that could contribute to cervical ripening. Full-thickness membranes were obtained from patients at caesarean section in the presence or absence of labour. Membranes were sampled from three regions: close to the site of placental attachment, in the region of the internal cervical os, and from a ‘middle’ area between these two. PGDH activities (sum of PGF 2α to 15-keto PGF 2α and 13, 14-dihydro 15-keto PGF 2α conversion) and immunoreactivity varied appreciably between the three regions. PGDH activity was highest in chorion in the cervical region of patients not in labour, but was significantly lower in the chorion from membranes taken closest to the internal os of patients in labour. Loss of PGDH activity was not attributable to a diminution in the number of trophoblast cells in this area. We conclude that regional loss of PGDH in the fetal membranes (chorion) at the lower uterine segment occurs with labour. Reduced PG catabolism could facilitate local generation of bioactive PG at this site for cervical effacement.

The ovarian regulation of ovulation in teleost fish

Ovulation is the release of a mature oocyte from its follicle wall enclosure in the ovary. This process requires the separation of the oocyte from the granulosa layer, the rupture of the follicle wall and the active expulsion of the oocyte through the rupture site. Results of experiments on various vertebrates, including fish, have shown that the control of these processes may involve the cooperative action of a number of ovarian regulators including proteases, protease inhibitors, progestational steroids, eicosanoids, catecholamines and vasoactive peptides. We have used two teleost models, the brook trout (Salvelinus fontinalis) and the yellow perch (Perca flavescens) to study the mechanism and control of ovulation in fish. Using subtractive cDNA cloning, a family of ovarian and ovulation specific mRNAs (“TOPs” = trout ovulatory proteins) was isolated from the brook trout ovary. These mRNAs have not previously been observed in the ovary of any vertebrate species; however, the proteins they code for have significant sequence homology to a group of mammalian protease inhibitors called antileukoproteinases. These inhibitors have been isolated from several mammalian mucosal tissues and their function may be to protect the mucosal lining from nonspecific degradation by proteases released from infiltrating leukocytes. The ovarian proteins encoded by the TOP mRNAs have now been characterized by Western blotting using antibodies derived against recombinant TOPs. Given the similarity of TOPs to antileukoproteinases, one function of TOPs may be to regulate proteolysis at the time of ovulation. In yellow perch, the maturational steroid, 17α,20β-dihydroxy-4-pregnen-3-one (17,20-PG), stimulates both germinal vesicle breakdown and ovulation in vitro. The stimulation of ovulation can be blocked by indomethacin, a prostaglandin endoperoxide synthase inhibitor. Thus, it appears that 17,20-PG acts through the production of an eicosanoid that is most likely a primary prostaglandin. This hypothesis is further supported by the observations that (1) a direct correlation exists between indomethacin levels that block ovulation and those that block primary prostaglandin synthesis in the ovary; (2) ovulation can be restored in indomethacin-blocked incubates with primary prostaglandins; (3) PGF levels increase at the time of ovulation in incubations of yellow perch follicles stimulated with 17,20-PG; and (4) the stimulation of PGF by steroids in the ovary is specific for 17,20-PG. Finally, 17,20-PG-stimulated ovulation and follicular prostaglandin synthesis requires the close interaction of extrafollicular tissue and other follicle wall layers.

Immunoreactive 15-hydroxyprostaglandin dehydrogenase (PGDH) is reduced in fetal membranes from patients at preterm delivery in the presence of infection

Previously we reported that the proportion of trophoblast cells that were immunopositive for 15-OH prostaglandin dehydrogenase (PGDH) in the chorionic membranes was reduced in women in preterm labour without infection, compared with women at term, but was not altered in preterm labour patients with an underlying infective process. Subsequently, we found that PGDH activity and PGDH mRNA were significantly lower in membranes of this latter group of patients than in women at preterm labour without infection or at term. To resolve this issue we used immunohistochemistry to examine the distribution and frequency of immunoreactive (ir)-PGDH positive cells in full-thickness fetal membranes in patients at preterm labour in the presence or absence of infection. Trophoblast and decidual stromal cells were identified using antibodies against cytokeratin and vimentin, respectively. There was considerable variation in the number of chorionic trophoblast cells that were positive for it-PGDH, but in some patients there was little or no it-PGDH staining, and this was associated with loss of trophoblast cells from the tissue. The mean intensity and number of it-PGDH positive cells was significantly lower in membranes from patients in preterm labour with infection than in idiopathic preterm labour at which the diagnosis of infection was not made. We conclude that in the setting of preterm labour with infection there may be loss of trophoblast cells from membranes, with corresponding reduction in the number of ir-PGDH positive cells. Loss of PGDH activity removes the initial step in activating primary prostaglandins, which are then able to pass unmetabolized to the decidua and myometrium, and contribute to the stimulus to preterm birth.

Local Amplification of Platelet Function by 8-Epi Prostaglandin F2α Is Not Mediated by Thromboxane Receptor Isoforms

8-epi-Prostaglandin (PG) F2alpha may be formed by cyclooxygenases 1 and 2 or by a free radical catalyzed process as an isoprostane. Concentrations of 8-epi-PGF2alpha in the range 1 nM to 1 microM induce a dose-dependent increase in platelet shape change, in calcium release from intracellular stores [Ca2+]iand in inositol phosphates; it also causes irreversible platelet aggregation, dependent on thromboxane generation, when incubated with subthreshold concentrations of ADP, thrombin, collagen, and arachidonic acid. Much higher concentrations of 8-epi-PGF2alpha (10-20 microM) alone induce weak, reversible aggregation. Although these effects are prevented by pharmacological thromboxane receptor antagonists, they are unlikely to be mediated by thromboxane receptors. Thus, 8-epi-PGF2alpha does not compete for binding at the stably expressed placental or endothelial isoforms of the thromboxane receptor or for binding of thromboxane ligands to human platelets. Furthermore, the response to 8-epi PGF2alpha exhibits structural specificity versus 8-epi PGF3alpha and PGF2alpha. Concentrations in the range that evoke its effects on platelets do not desensitize the aggregation response stimulated by thromboxane or PGH2 analogs. Unlike primary prostaglandins, which are rapidly metabolized to inactive products, 8-epi PGF2alpha circulates in plasma. However, the systemic concentrations found in healthy volunteers (median 48 pmol/liter) and in patients with hepatic cirrhosis (median 147 pmol/liter), a syndrome of oxidant stress in vivo, fall well below those which modulate platelet function. 8-Epi PGF2alpha may amplify the response to platelet agonists in syndromes where oxidant stress and platelet activation coincide. Despite blockade by thromboxane antagonists, 8-epi PGF2alpha does not activate either of the thromboxane receptor isoforms described in platelets. Activation of a distinct receptor would be consistent with the enzymatic formation of 8-epi PGF2alpha by cyclooxygenases. However, incidental activation of such a receptor by systemic concentrations of 8-epi PGF2alpha is unlikely to occur, even in syndromes of excessive free radical generation in vivo.

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.