The effect of estradiol on COX-2, EP2, and EP4 mRNA expression and the extracellular matrix in the cervix of the hypogonadotrophic, ovariectomized ewe

There is a degree of cervical relaxation in the ewe at estrus that is regulated by changes in prostaglandin synthesis, prostaglandin receptor expression, and changes in the cervical extracellular matrix. It is likely that these are regulated by changes in periovulatory hormones, particularly estradiol. This study determined the effect of estradiol benzoate on the mRNA expression of cyclooxygenase-2 (COX-2) and the prostaglandin E receptors EP₂ and EP₄, the concentration of cervical hyaluronan, and the proportion of smooth muscle and collagen in the cervix of the hypogonadotrophic ovariectomized ewe (Ovis aries). Ovariectomized hypogonadotrophic ewes were given 100 μg estradiol benzoate, and their cervices were collected 0, 24, and 48 h thereafter to determine the expression of cervical COX-2, EP₂, and EP₄ mRNA by in situ hybridization, the concentration of hyaluronan by ELISA, and the proportion of smooth muscle and collagen by Masson's trichrome staining. Estradiol benzoate increased the mRNA expression of COX-2 and EP₄ within 24 h after treatment (P < 0.05), whereas EP₂ mRNA, hyaluronan, and the ratio of smooth muscle to collagen did not change within 48 h after treatment. The COX-2, EP₂, and EP₄ mRNA expression were greatest in the smooth muscle layers (P < 0.05) and least in the luminal epithelium (P < 0.05). In conclusion, we inferred that estradiol regulates cervical COX-2 and EP₄ mRNA expression and may regulate cervical relaxation via the synthesis of prostaglandin E₂ and activation of the PGE₂ receptors EP₂ and EP₄.     

Synthesis and evaluation of a potent,well-balanced EP2/EP3 dual agonist

A highly potent and well-balanced dual agonist for the EP₂ and EP₃ receptors is described. Optimization of the lead compound was accomplished in consideration of the relative agonist activity against each EP subtype receptor and the pharmacokinetic profile. As the result, 2-[(2-{(1R,2R)-2-[(1E,4S)-5-cyclopentyl-4-hydroxy-4-methyl-1-penten-1-yl]-5-oxocyclopentyl}eth-yl)thio]-1,3-thiazole-4-carboxylic acid (10) showed excellent potency (human EC₅₀ EP₂?=?1.1?nM, EP₃?=?1.0?nM) with acceptable selectivity over the EP₁ and EP₄ subtypes (>2000-fold). Further fine-tuning of compound 10 led to identification of ONO-8055 as a clinical candidate. ONO-8055 was effective at an extremely low dose (0.01?mg/kg, po, bid) in rats, and dose-dependently improved voiding dysfunction in a monkey model of underactive bladder (UAB). ONO-8055 is expected to be a novel and highly promising drug for UAB.     

The mRNA expression of prostaglandin E receptors EP2 and EP4 and the changes in glycosaminoglycans in the sheep cervix during the estrous cycle

Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E₂ acts via prostaglandin E receptors EP₁ to EP₄, and EP₂ and EP₄ stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP₂ and EP₄ mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during, and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP₂ and EP₄ was determined by in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was investigated by ELISA. The expression of EP₂ mRNA was greatest prior to the LH surge (P = 0.02), although EP₂ and EP₄ were expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased prior to the LH surge (P < 0.05). Cervical EP₂ mRNA expression changed throughout the estrous cycle and was greatest prior to the LH surge. We propose that prostaglandin E₂ binds to EP₂ and EP₄ stimulating hyaluronan synthesis, which may cause remodeling of the cervical extracellular matrix, culminating in cervical relaxation.     

EP2 receptor activation by prostaglandin E2 leads to induction of HO-1 via PKA and PI3K pathways in C6 cells

Recently we proposed that COX-2 induction precedes expression of HO-1 in ischemic preconditioned rat brain. In the current study, we investigated the molecular mechanism by which prostaglandin E₂, one of COX-2 metabolites, induces HO-1 in rat C6 brain cells. We demonstrated that concentration of PGE₂ increased HO-1 expression in C6 cells in vitro. The effects of PGE₂ were mimicked by PGE₂ receptor EP₂ agonists, 11-deoxy PGE₂, and cAMP analog, dibutyl-cAMP. HO-1 expression by PGE₂ was inhibited by LY294002, PI3K inhibitor and H89, PKA inhibitor. The EP₂-specific antagonist, AH8006 also inhibited PGE₂-mediated HO-1 expression in a concentration-dependent manner. Finally, PGE₂ inhibited GOX-induced apoptosis as assayed by FACS analysis or DNA strand breaks assay, and this cell death was reversed by ZnPPIX, HO-1 inhibitor. In addition to HO-1 induction, PGE₂ also increased phosphorylation of Bad by PKA- and PI3K-depednent manner. Taken together, we conclude that PGE₂ induces HO-1 protein expression through PKA and PI3K signaling pathways via EP₂ receptor in C6 cells. The induction of HO-1 along with increase of p-Bad by PGE₂ is responsible for anti-apoptosis against oxidant stress.     

The prostaglandin transporter PGT transports PGH2

Prostaglandin H₂ not only serves as the common precursor of all other PGs, but also directly triggers signals (e.g. platelet aggregation), depending on its location and translocation. The prostaglandin carrier PGT mediates the transport of several prostanoids, such as PGE₂, and PGF_2α. Here we used PGT in the plasma membrane as a model system to test the hypothesis that PGT also transports PGH₂. Using wild-type and PGT-expressing MDCK cells, we show that PGH₂ uptake is mediated both by simple diffusion and by PGT. The PGH₂ influx permeability coefficient for diffusion is (5.66 ± 0.63) × 10⁻⁶ cm/s. The kinetic parameters of PGH₂ transport by PGT are K_m = 376 ± 34 nM and V_max = 210.2 ± 11.4 fmol/mg protein/s. PGH₂ transport by PGT can be inhibited by excess PGE₂ or by a PGT inhibitor. We conclude that PGT may play a role in transporting PGH₂ across cellular membranes.     

EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation

Prostaglandin-E₂ (PGE₂) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE₂ mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP₁, EP₂, EP₃ and EP₄. The EP₃ prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP₃ receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP_3-Ia, EP_3-II or EP_3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE₂ strongly stimulated the phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP_3-Ia isoform was minimal and only occurred at μM concentrations of PGE₂. Furthermore, the mechanisms of the PGE₂ mediated phosphorylation of ERK 1/2 by the EP_3-II and EP_3-III isoforms were different. Thus, PGE₂ stimulation of ERK 1/2 phosphorylation by the EP_3-III isoform involves activation of a Gα_i/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP_3-II isoform it involves activation of a Gα_i/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP_3-II and EP_3-III prostanoid receptor isoforms.     

Synthesis of new organotitanium (IV) complexes Cp2Ti(SeR)2 and Cp2TiCl(SeR) via tandem reactions involving `Cp2Ti' intermediate. Crystal structures of Cp2TiCl(SeR) (R=p-ClC6H4, p-BrC6H4)

A simple and convenient route for synthesizing organotitanium (IV) complexes with a general formula Cp₂Ti(SeR)₂ or Cp₂TiCl(SeR) has been developed. This synthetic route includes reduction of Cp₂TiCl₂ with Mg and an in situ treatment of the intermediate `Cp₂Ti' with diselenides RSeSeR. Interestingly, while the route involving reaction of Cp₂TiCl₂, Mg and RSeSeR in a molar ratio of 1:1:1 produced Cp₂Ti(SeR)₂, (1-5, R=α-C₁₀H₇, o-MeC₆H₄, m-MeC₆H₄, p-ClC₆H₄, p-BrC₆H₄) in 91-97% yields, the route involving reaction of Cp₂TiCl₂, Mg and RSeSeR in a molar ratio of 1: 0.5: 0.5 afforded Cp₂TiCl(SeR) (6-7, R=p-ClC₆H₄, p-BrC₆H₄) in 70% and 92% yields, respectively. 1-7 are new and have been characterized by elemental analysis and spectroscopy, as well as by X-ray diffraction analysis for 6 and 7. A possible pathway for production of these two types of organotitanium (IV) complexes, mainly depending upon the molar ratio of the starting materials, are briefly discussed.     

The EP1 subtype of prostaglandin E2 receptor: Role in keratinocyte differentiation and expression in non-melanoma skin cancer

We have previously demonstrated that the EP₁ subtype of PGE₂ receptor is expressed in the differentiated compartment of normal human epidermis and is coupled to intracellular calcium mobilization. We therefore hypothesized that the EP₁ receptor is coupled to keratinocyte differentiation. In in vitro studies, radioligand binding, RT-PCR, immunoblot and receptor agonist-induced second messenger studies demonstrate that the EP₁ receptor is up-regulated by high cell density in human keratinocytes and this up-regulation precedes corneocyte formation. Moreover, two different EP₁ receptor antagonists, SC51322 and AH6809, both inhibited corneocyte formation. SC51322 also inhibited the induction of differentiation-specific proteins, cytokeratin K10 and epidermal transglutaminase. We next examined the immunolocalization of the EP₁ receptor in non-melanoma skin cancer in humans. Well-differentiated SCCs exhibited significantly greater membrane staining, while spindle cell carcinomas and BCCs had significantly decreased membrane staining compared with normal epidermis. This data supports a role for the EP₁ receptor in regulating keratinocyte differentiation.     

Tryptase activates isolated adult cardiac fibroblasts via protease activated receptor-2 (PAR-2)

Protease activated receptor-2 (PAR-2) derived cycloxygenase-2 (COX-2) was recently implicated in a cardiac mast cell and fibroblast cross-talk signaling cascade mediating myocardial remodeling secondary to mechanical stress. We designed this study to investigate in vitro assays of isolated adult cardiac fibroblasts to determine whether binding of tryptase to the PAR-2 receptor on cardiac fibroblasts will lead to increased expression of COX-2 and subsequent formation of the arachodonic acid metabolite 15-d-Prostaglandin J₂ (15-d-PGJ₂). The effects of tryptase (100 mU) and co-incubation with PAR-2 inhibitor peptide sequence FSLLRY-NH₂ (10^-6M) on proliferation, hydroxyproline concentration, 15-d-PGJ₂ formation and PAR-2/COX-2 expression were investigated in fibroblasts isolated from 9 week old SD rats. Tryptase induced a significant increase in fibroproliferation, hydroxyproline, 15-d-PGJ₂ formation and PAR-2 expression which were markedly attenuated by FSLLRY. Tryptase-induced changes in cardiac fibroblast function utilize a PAR-2 dependent mechanism.     

Halo-complexes of titanium(III): The thermochromic behaviour of [NBu4][TiCl4(THF)2]

TiCl₃(thf)₃ reacts with ACl (A = NBu₄, PPN; PPN = Ph₃PNPPh₃) in dichloromethane solution, affording the compounds A[TiCl₄(thf)₂] (A = NBu₄, 1; A = PPN, 2). Compound 1, dissolved in CH₂Cl₂, exhibits thermochromic behaviour which has been the subject of variable-temperature UV-Vis investigations.     

Characterization of Brain Synaptic Vesicle Phospholipase A2 Activity and Its Modulation by Calmodulin, Prostaglandin E2, Prostaglandin F2α, Cyclic AMP, and ATP

Brain synaptic vesicle phospholipase A2 (PLA2) activity was characterized. It is Ca2+-dependent and has a pH optimum of 9.0. The enzyme has a Km of 60 microM and a Vmax of 2.0 nmol/mg/h. Calmodulin, prostaglandin F2 alpha, and cAMP, and ATP all increased the Vmax of the enzyme. Prostaglandin E2 inhibited the Vmax in the presence or absence of calmodulin. Light-scattering techniques in conjunction with phase-contrast and electron microscopy demonstrated that an increase in Vmax of PLA2 was correlated with synaptic vesicle aggregation, lysis, and possible fusion. In vitro synaptic vesicle-vesicle association that was stimulated by conditions that increased PLA2 activity could be diminished when synaptic vesicles were preincubated with PLA2 inhibitors. It is suggested that endogenous synaptic vesicle PLA2 activity may be an important mechanism underlying Ca2+-mediated neurotransmitter release.     

Applications and limitations of measurement of 15-keto,13,14-dihydro prostaglandin E2 in human blood by radioimmunoassay

It has been anticipated that the inherent limitations of radioimmunoassays for prostaglandin E (PGE) would be obviated by assays for its major circulating metabolite, 15-keto, 13,14-dihydro PGE₂ (KH₂-PGE₂) which has a longer half-life in blood. We examined the effects of PGE₂ infusion and alterations in lipolysis , and of clotting, prolonged storage and hemolysis , on KH₂-PGE₂ immunoreactivity in unextracted human plasma and serum samples. Indeed KH₂-PGE₂ levels rose several hundred fold during infusions of PGE₂ at doses which cause little or no increment in peripheral PGE levels. During stimulation of lipolysis by infusions of epinephrine, apparent KH₂-PGE₂ levels rose five-fold. However, the dilution curve of plasma obtained during stimulation of lipolysis was not parallel to the standard curve; furthermore, apparent KH₂-PGE₂ levels were correlated strongly with free fatty acid (FFA) levels, suggesting that FFA's cross-reacted in the RIA weakly but significantly due to their very high molar concentration in blood. Clotting and prolonged storage of samples, but not hemolysis, also caused marked apparent increments in KH₂-PGE₂ levels. Competition curves using dilutions of such samples were again not parallel to the standard curves in plasma or buffer, but resembled dilution curves of samples containing high levels of FFA. These results suggest that handling of human blood samples for KH₂-PGE₂ measurement must be carefully standardized to avoid significant artifacts which presumably are due in part to fatty acids released from triglyceride stores or from disrupted membrane phospholipids . Unextracted plasma appears to be unsatisfactory for use in this RIA.     

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.     

Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl}acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain

A novel series of EP₄ agonists and antagonists have been identified, and then used to validate their potential in the treatment of inflammatory pain. This paper describes these novel ligands and their activity within a number of pre-clinical models of pain, ultimately leading to the identification of the EP₄ partial agonist GSK726701A.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Structural insights into PDZ-mediated interaction of NHERF2 and LPA2, a cellular event implicated in CFTR channel regulation

The formation of CFTR–NHERF2–LPA₂ macromolecular complex in airway epithelia regulates CFTR channel function and plays an important role in compartmentalized cAMP signaling. We previously have shown that disruption of the PDZ-mediated NHERF2–LPA₂ interaction abolishes the LPA inhibitory effect and augments CFTR Cl⁻ channel activity in vitro and in vivo. Here we report the first crystal structure of the NHERF2 PDZ1 domain in complex with the C-terminal LPA₂ sequence. The structure reveals that the PDZ1–LPA₂ binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four LPA₂ residues contributing to specific interactions. Comparison of the PDZ1–LPA₂ structure to the structure of PDZ1 in complex with a different peptide provides insights into the diverse nature of PDZ1 substrate recognition and suggests that the conformational flexibility in the ligand binding pocket is involved in determining the broad substrate specificity of PDZ1. In addition, the structure reveals a small surface pocket adjacent to the ligand-binding site, which may have therapeutic implications. This study provides an understanding of the structural basis for the PDZ-mediated NHERF2–LPA₂ interaction that could prove valuable in selective drug design against CFTR-related human diseases.     

The use of oral prostaglandin E2 in the management of intrauterine fetal death

12 otherwise healthy patients with intrauterine fetal death 1 to 6 weeks earlier were treated with oral prostaglandin E₂. 9 of the 12 patients delivered within 48 hours after treatment began. 2 others delivered within 48 hours after unsuccessful treatment ceased. In a third patient the cervix relaxed after treatment, and the uterine contents were removed by curettage. No serious complications, such as hemorrhage occurred. The uterus seemed surprisingly responsive to oral prostaglandin E₂ in cases of intrauterine fetal death.     

Controlled trial of induction of labor by vaginal suppositories containing prostaglandin E2

A group of 84 women at 39 – 43 weeks of pregnancy were randomly allocated to a blind trial of induction of labor with vaginal suppositories containing inert material or either 0.2 mg or 0.4 mg of prostaglandin E₂. The suppositories were self-administered every two hours during waking hours on two successive days until labor started or 15 had been used. Side-effects were absent. Labor was established within 48 hr of insertion of the first suppository in 9.3% of control patients, 65.4% of those treated with 0.2 mg PGE₂ and 85.7% of those treated with 0.4 mg PGE₂. The mean Apgar scores in the three groups were the same. The mean total dose of PGE₂ were 2.0 mg (0.2 mg group) and 2.3 mg (0.4 mg group). It is concluded that vaginal PGE₂ is an effective and acceptable method of inducing labor at term.     

Ibuprofen reduces zebrafish PGE2 levels but steroid hormone levels and reproductive parameters are not affected

Prostaglandins are important regulators of reproductive function in fish. Analgesics like aspirin and ibuprofen are prostaglandin inhibitors and have been detected in freshwater systems at ng/L–μg/L levels. We investigated whether ibuprofen would affect prostaglandin and sex steroid hormone levels in adult zebrafish (Danio rerio) and if expression levels of genes involved in steroidogenesis and prostaglandin synthesis were affected. Zebrafish were exposed to moderate concentrations of ibuprofen (21, 201 or 506 μg/L) for 7 days in a semi-static test system. Ibuprofen concentrations were close to nominal levels and decreased by a maximum of 12–13% over 24 h. Prostaglandin E₂ (PGE₂) levels in whole body homogenates of males and ovaries of females decreased in a monotonic dose–response relationship whereas male 11-ketotestosterone levels and ovarian 17β-estradiol levels remained unchanged. Ibuprofen did not have an influence on vitellogenin levels, female gonadosomatic index or cumulative egg production and no dose–response relationship in ovarian and testicular expression levels of the investigated genes was observed. This study shows that ibuprofen reduces PGE₂ levels in male and female zebrafish but has no consistent effects on other investigated reproductive parameters.