Selected properties of [3H]prostaglandin E1 binding to dispersed bovine luteal cells.

Selected properties of [3H]prostaglandin (PG) E1 binding to collagenase dispersed bovine luteal cells were studied and compared with those observed in luteal plasma membranes. [3H]-PGE1 specific binding to a relatively homogeneous population of luteal cells was a rapid (K1 = 4.2 X 10(5) M-1 .sec-1), reversible (K-1 = 3.9 X 10(-3) sec-1), saturable and specific process at 38 degrees C. The binding was homogeneous with an apparent dissociation constant of 2.4 nM and 1.8 X 10(5) receptors per cell. The presence of increasing amounts of unlabeled PGs inhibited [3H]PGE1 binding in a dose-dependent manner. The potency order for this inhibition of binding was: PGE 2 greater than PGE1, (15S)-15-methyl-PGE2 methyl ester greater than PGF2alpha greater than PGF1alpha greater than other PGs, PGE, PGF metabolites and PGF analogs. Other than the homogeneous nature of [3H]PGE1 binding and the greater effectiveness of PGE2 compared to PGE1 in cells, the rest of the properties of [3H]PGE1 binding to cells were in excellent agreement with those observed in plasma membranes.     

Prostaglandin E2 receptor binding and action in human fat cells

The prostaglandin E2 (PGE2) receptor in human adipocytes was identified by the use of [3H]PGE2. The receptor binding at physiological temperature and pH was specific, saturable, and slowly reversible. Half-maximal displacement for [3H]PGE2 binding occurred with 2.5 nmol/liter. Half-maximal inhibition of isoproterenol-induced lipolysis was achieved at a concentration of PGE2 of 3.8 nmol/liter and half-maximal inhibition of basal lipolysis was achieved at a concentration of PGE2 of 0.9 nmol/liter. The order of potency for prostaglandin inhibition of receptor binding and antilipolytic effect was the same, with PGE2 greater than PGF2 alpha much greater than arachidonic acid. Scatchard analysis of the binding data revealed a nonlinear plot indicating the existence of two or more binding sites with different affinities. The binding sites of high affinity had an equilibrium constant (Kd) of 2 nmol/liter and a total binding capacity of 58 fmol/10(6) adipocytes which corresponds to about 33,000 binding sites per adipocyte. The binding sites of low affinity had a Kd of 56 nmol/liter and a total binding capacity of 700 fmol/10(6) adipocytes. We conclude that [3H]PGE2 binds to receptors in isolated human adipocytes and that their antilipolytic effects are mediated by this binding.     

Specific binding of prostaglandin F2 alpha to membranes of rat corpora lutea.

Prostaglandin F2 alpha (PGF2 alpha) binds specifically to a partially purified membrane preparation from rat corpora lutea. The high affinity, low capacity binding component asa a Kd = 4.7 nM and has a capacity of 0.38 pmol/mg protein. Binding kinetics were temperature-dependent with an association rate constant of 2.5 x 10(5) 1/mol-sec and a dissociation rate constant of 4.3 x 10(-4) sec-1 at 22 degrees C. Little competition for binding was shown by other prostaglandins and prostaglandin metabolites; the PGF2 alpha analogue ICI 81008 (16-m-trifluoromethylphenyl-prostaglandin F2 alpha) showed a binding affinity similar to that of PGF2 alpha. The specific binding of PGF2 alpha to luteal cell membranes was confirmed by electron microscopy using a ferritin--PGF2 alpha conjugate. Ferritin--PGF2 alpha was found predominantly on luteal cell surfaces; little binding occurred on other types of cells present. These data demonstrate specific binding of PGF2 alha to rat luteal membranes. It is suggested that the luteolytic action of PGF2 alpha in the rat may be receptor-mediated.     

Specific receptors for epidermal growth factor in human bone tumour cells and its effect on synthesis of prostaglandin E2 by cultured osteosarcoma cell line

Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E2, a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125I-labelled EGF: the apparent dissociation constant was approximately 4-10 X 10(-10) M and the maximal binding capacity was 50 000-80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE2 into medium, while no significant amount of PGE2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE2 production in HOSO line in a dose-dependent manner (0.5-50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGF2 alpha, PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 or G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE2-mediated process in human osseous tissues.     

Beta-adrenergic regulation of prostaglandin E2 receptors in human and rat adipocytes

Incubation of intact human and rat adipocytes with isoproterenol (10(-6) M) inhibits the specific binding of [3H] prostaglandin E2 (PGE2) by about 25% in human adipocytes and 50% in rat adipocytes. Scatchard analysis of [3H]PGE2 binding demonstrated that the isoproterenol-induced decrease in receptor activity may be due to a decrease in the apparent number of PGE2-binding sites, while the receptor affinity was unaltered. The inhibitory effect of isoproterenol on [3H]PGE2 binding was already seen after 10 min of isoproterenol treatment, and the maximal effect was obtained after 30-60 min. Half-maximal inhibition of binding occurred at a concentration of 5 X 10(-8) M isoproterenol. The effect of isoproterenol could be mimicked by epinephrine, theophylline, and (Bu)2-cAMP, indicating that elevated levels of cAMP are the common mechanism by which these agents affect PGE2 binding. Furthermore, the isoproterenol-induced inhibition of PGE2 binding was completely blocked by propranolol. The effects of some FFA and indomethacin were studied on PGE2 receptor binding, too. From the results of the latter studies, we suggest that endogeneously released arachidonic acid could account for some of the reduction in PGE2 binding. In conclusion, a beta-adrenergic receptor-mediated cAMP-dependent mechanism for the regulation of PGE2 receptor binding is demonstrated in both human and rat adipocytes.     

Topography of human uterine prostaglandin E and F2 alpha receptors and their profiles during pathological states

The topography of prostaglandin (PG) E and F2 alpha receptors in uteri of premenopausal women was investigated by dividing uteri into six equal longitudinal strips and further dividing each strip into approximately 1-cm segments. Tissue for determination of smooth muscle content using the Trichrome stain was taken from each section, and the remainder was homogenized for binding studies with 3H-labeled PGs. The [3H] PGE1 binding (mean, 41.5 fmol/mg protein; range, 23.1-58.3) was about 8-fold greater in the fundus than [3H]PGF2 alpha binding (mean, 4.8 fmol/mg protein; range, 1.3-13.0), and this trend was found in most uterine sections. The binding of both 3H-labeled PGs decreased from fundus to cervix, and this decrease was similar to the decrease in smooth muscle content. Scatchard analysis revealed apparent dissociation constants (Kds) of 1.4 and 76 nM and apparent specific binding capacities (Ns) of 25 and 488 fmol/mg protein for [3H]PGE2, and Kd values of 11.5 and 81 nM and Ns values of 19.4 and 58 fmol/mg protein for [3H]PGF2 alpha in the uterine fundus. The Kd values for [3H]PGE2 were similar in other sections of the uterus, but the Ns values were smaller in the lower uterine body and cervical end. While the phase of the menstrual cycle did not influence [3H]PG binding, the diagnosis of abnormal uterine bleeding compared to dysmenorrhea was associated with an increase in [3H]PGE1 binding (P less than 0.05).     

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.     

Potentiation of prostaglandin E2-induced release of LH by the prostaglandin analogue, 7-oxa-13-prostynoic acid

The prostaglandin (PG) analogue 7-oxa-13-prostynoic acid (7-OPA) was infused into a lateral ventricle of the brain of adult male rats and the effect of the analogue on the subsequent stimulation of LH release by intraventricular infusion of PG's was determined. Pretreatment of the animals with 44- 132 micrograms of 7-OPA potentiated the stimulatory effect of 2 micrograms PGE2 on the release of LH but the analogue alone had no effect on the hormone secretion. The minimal effective dose of PGE2 was determined to be within the range 0.01-0.05 micrograms and it was found that priming with 132 micrograms of 7-OPA caused a formerly sub-threshold dose (0.01 micrograms) of PGE2 to become an effective stimulus for the release of LH. In contrast to its potentiating effect on PGE2-induced LH release 7-OPA did not alter the stimulatory action of PGF2 alpha (2 micrograms) on the secretion of LH. 7-OPA had no effect on LRH-induced release of LH indicating that PG analogue acts at a suprapituitary site to enhance PGE2-induced LH release. The potentiating effect of 7-OPA may be exerted at a binding site for PGE2 in the brain and the results suggest the existence of a different binding site for PGF2 alpha. The possibility also exists that 7-OPA inhibit metabolic inactivation of PGE2.     

Simultaneous infusion of prostaglandin E2 antagonizes the luteolytic action of prostaglandin F2alpha in vivo

Corpora lutea of ewes bearing ovarian autotransplants were infused for 4 h with prostaglandin F2alpha (PGF2alpha) (10 microng/h), PGF2alpha+PGE2 (10microng/h of each), PGE2 (10 microng/h) or saline on day 10 of the cycle. Ovarian venous blood obtained before, during, and up to 12 h after the infusion period, was assayed for progesterone. Prostaglandin F2alpha produced an immediate, rapid and sustained decline in progesterone secretion, but infusion of PGE2 together with PGF2alpha prevented the decline until after the infusion. Progesterone secretion was unaffected by infusion of PGE2 alone. Oestrous behaviour was observed in four out of seven animals infused with PGF2alpha but in only one out of six infused with PGF2Alpha+PGE2. None of the animals infused with PGE2 alone or saline only came into heat.     

Noradrenaline and prostaglandin E2 stimulate LH-RH release from rat median eminence through distinct 1-alpha-adrenergic and PGE2 receptors

Noradrenaline (NA) and prostaglandin (PG) E2 produced a dose-related stimulation of luteinizing hormone releasing hormone (LH-RH) release from incubated median eminence of adult male rats, with ED50 values of 6.10(-7) and 8.10(-8) M, respectively. The effects of some adrenoceptor agonists (10(-5) M) on LH-RH release were tested: only phenylephrine (alpha 1-agonist) stimulated LH-RH release; clonidine (alpha 2 greater than alpha 1-agonist) and isoproterenol (beta-agonist) were ineffective. Adrenoceptor antagonists (10(-6) M) were also tested: prazosin (alpha 1-antagonist) and phentolamine (alpha 1/alpha 2-antagonist) almost completely suppressed the enhanced release of LH-RH induced by NA. In contrast, neither yohimbine (alpha 2-antagonist) nor propranolol (beta-antagonist) altered this effect of NA. When tested alone, no significant effect was obtained on basal LH-RH release with any of the antagonists tested. Moreover, at concentrations that blocked the stimulation produced by NA, the adrenoceptor antagonists did not alter the effect of PGE2. Among seven PGs tested at 10(-6) M, only PGE2, PGE1, PGA2, and 16,16-dimethyl PGE2 significantly enhanced LH-RH secretion. 8-iso PGE2 weakly stimulated LH-RH secretion, whereas PGF2 alpha and PGD2 were ineffective. A direct correlation existed between the potency of these compounds to modify LH-RH secretion and to inhibit specific [3H]-PGE2 binding to hypothalamic membranes. In conclusion, these results suggest that the stimulation of LH-RH from median eminence induced by NA and PGE2 involves the activation of an alpha 1-adrenergic receptor and a PGE2 receptor, respectively.     

Studies on the prostaglandin E-2-9-ketoreductase mediated production of prostaglandin F-2 alpha and its metabolism in cultured rabbit luteal cells

Luteal cells were isolated from pseudopregnant rabbits on days 3, 6, 9, 12 and 15 post ovulation. Prostaglandin concentration and the activities of the enzymes prostaglandin E-2-9-ketoreductase and prostaglandin-15-hydroxydehydrogenase were determined. Luteal cells from day 7 and 12 of pseudopregnancy were maintained in culture for 24 h and then exposed to a mixture of [1-14C]PGE2 (70 mumol/l) in the presence or absence of estradiol-17 beta. After a 24 h incubation period, the culture medium was adjusted to pH 3.5, immediately extracted and analysed for PG. Cultured luteal cells were able to convert exogenously applied PGE2 to PGF2 alpha and to metabolize both PGs. Primary PGs as well as their metabolites 15-keto PGE2, 15-keto PGF2 alpha, 13,14-dihydro-15-keto PGE2, and 13,14 dihydro-15-keto PGF2 alpha were detected in the culture medium and in the cells. The addition of estradiol-17 beta together with PGE2 caused a significant reduction in the PGE2-9-ketoreductase mediated PGF2 alpha synthesis, whereas the metabolism of PGE2 remained unchanged. This inhibitory effect of estradiol-17 beta was dose-dependent on day 12 of pseudopregnancy. The results demonstrate that isolated rabbit luteal cells are able to synthesize and metabolize the luteolytic factor PGF2 alpha. Whether the inhibitory effect of estradiol-17 beta may have any physiological relevance has to be examined in further studies.     

The effects of lipopolysaccharide and interleukins-1alpha, -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium

Up-regulation of endometrial oxytocin receptor (OTR) expression followed by an increase in pulsatile endometrial prostaglandin (PG) F(2alpha) secretion causes luteolysis in cattle. Inhibition of luteolysis is essential for the maternal recognition of pregnancy but also occurs in association with endometritis. The factors regulating OTR expression at this time are unclear. The OTR gene promoter region contains binding elements for acute phase proteins but their function has not been established. This study investigated the effects of various cytokines on OTR expression and on PGF(2alpha) and PGE(2) production in explant cultures of bovine endometrium. Endometrium was collected in the late luteal phase (mean day of cycle 15.4+/-0.50) or early luteolysis (mean day of cycle 16.4+/-0.24) as determined by the initial concentration of endometrial OTR. Explants were treated for 48 h with: (i) lipopolysaccharide (LPS) and/or dexamethasone (DEX), (ii) ovine interferon-tau (oIFN-tau), or (iii) human recombinant interleukin (IL)-1alpha, -2 or -6. OTR mRNA was then measured in the explants by in situ hybridisation and the medium was collected for measurement of PGF(2alpha) and PGE(2) by RIA. LPS treatment stimulated production of PGF(2alpha), whereas DEX either alone or in combination with LPS was inhibitory to both PGF(2alpha) and PGE(2). Neither of these treatments altered OTR mRNA expression. oIFN-tau reduced OTR mRNA expression but stimulated production of both PGF(2alpha) and PGE(2). In endometrial samples collected in the late luteal phase, IL-1alpha, -2 and -6 all inhibited OTR mRNA expression, but IL-1alpha and -2 both stimulated PGF(2alpha) production. In contrast, when endometrium was collected in early luteolysis, none of the interleukins altered OTR expression or caused a significant stimulation of PGF(2alpha) production but IL-2 increased PGE(2). Neither IL-1alpha nor -2 altered OTR promoter activity in Chinese hamster ovary cells transfected with a bovine OTR promoter/chloramphenicol acetyl transferase reporter gene construct. In conclusion, the action of interleukins on both OTR mRNA expression and endometrial prostaglandin production alters around luteolysis. Pro-inflammatory interleukins suppress OTR expression in the late luteal phase, while LPS stimulates PGF(2alpha) without altering OTR mRNA expression. IL-I and -2 and LPS are therefore unlikely to initiate luteolysis but may cause raised production of PGF(2alpha) during uterine infection.     

Effects of prostaglandin F2alpha and E2 on the production of progesterone by human granulosa cells in tissue culture.

Human granulosa cells with differing steroidogenic potentials were cultured in vitro. The effects of prostaglandin F2alpha (PGF2alpha) and PGE2 on the progesterone output and viability of these cells were investigated. Prostaglandin F2alpha either alone or in combination with LH and FSH inhibited the production of progesterone over a wide range of concentrations (1-8000 ng/ml). However, the inhibitory effect of PGF2alpha was 200 times less effective when the cells were exposed to LH and FSH for 6 days before the addition of the prostaglandin. By contrast PGE2, at concentrations from 1 to 500 ng/ml, markedly stimulated the production of progesterone by granulosa cells, and this was not prevented by the addition of PGF2alpha. The degree of inhibition by PGF2alpha or stimulation by PGE2 was related to the biosynthetic capacity of the cells. These studies suggest that PGF2alpha may act directly on the adenylate cyclase system of human granulosa cells by blocking its activation by LH, and they demonstrate that functional regression of the luteal cell can be induced independently of the blood vascular system.     

Effects of prostaglandin F2 alpha on bone formation and resorption in cultured neonatal mouse calvariae: role of prostaglandin E2 production

Although most studies show that prostaglandin E2 (PGE2) is the most potent and effective of the prostanoids in bone, recent data in cell culture suggest that PGF2 alpha may have unique effects, particularly on cell replication. The present study was undertaken to compare the effects of PGF2 alpha and PGE2 in cultured neonatal mouse parietal bones by simultaneous measurement of bone resorption as release of previously incorporated 45Ca, bone formation as incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein, and DNA synthesis as incorporation of [3H]thymidine. PGF2 alpha was less effective than PGE2 as a stimulator of bone resorption, and its effects were partially inhibited by indomethacin and markedly inhibited by glucocorticoids. In contrast, the resorptive response to PGE2 was unaffected by indomethacin and only partially inhibited by cortisol. PGF2 alpha had little effect on bone formation, in contrast to the biphasic effect of PGE2, which inhibited labeling of CDP in the absence of cortisol and stimulated CDP labeling in the presence of cortisol. PGF2 alpha increased thymidine incorporation into DNA, but the effect was smaller than that of PGE2 and was inhibited by indomethacin. These observations suggested that PGF2 alpha might act in part by stimulating PGE2 production. By RIA, PGE2 concentrations were increased in the medium of bones treated with PGF2 alpha, and this increase was blocked by indomethacin. By HPLC, bones prelabeled with [3H]arachidonic acid showed an increase in labeled PGE2 release, and RIA showed an increase in PGE2 after PGF2 alpha treatment. These results indicate that PGF2 alpha is a relatively weak agonist in bone compared to PGE2 and that some of the effects of PGF2 alpha on bone resorption, formation, and cell replication may be mediated by an increase in endogenous PGE2 production.     

Allergen-stimulated release of prostaglandin E, F2 alpha by alveolar macrophages from patients with allergic asthma]

Bronchoalveolar lavage (BAL) was performed in healthy subjects (n = 12) and patients with bronchial asthma (n = 11). Between the two groups there was no significant difference in the total number of cells and the percentage of alveolar macrophages (AM), lymphocytes (L), neutrophils (N) and eosinophils (E) in BAL fluids. When AM were cultured in vitro and stimulated with dermatophagoides farinae (DPF) antigen, the amount of PGE released by AM was significantly increased in the asthmatics. When the asthmatics, AM were sensitized with specific IgE positive serum and stimulated with DPF, they released more PGE and PGF2 alpha than those when specific IgE negative serum was used (P < 0.01). The PGE/PGF2 alpha ratio was significantly decreased. There was positive correlation between the decreased value of PGE/PGF2 alpha and patients, MCH-PC20. The increase in the amount of PGE and PGF2 alpha released and the decrease of PGE/PGF2 alpha ratio might play an important role in the pathogenesis of allergic asthma.     

Prostaglandin E2 and prostaglandin F2 alpha biosynthesis in human gastric mucosa: effect of chronic alcohol misuse.

BACKGROUND AND AIMS: The results of experimental studies support the hypothesis that decreased prostaglandin production might play a part in the gastric mucosal injury induced by alcohol. In this study, it was investigated whether alcohol misuse impairs the synthesis of prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) in gastric mucosa. PATIENTS: Fifty six alcoholic patients and 66 subjects without alcohol misuse were included in the study. METHODS: Mucosal biopsy specimens were obtained from the antrum and body of the stomach. Maximal synthesis rates of PGE2 and PGF2 alpha were determined in the microsomal fraction of the biopsy specimens. RESULTS: The rates of synthesis of both prostaglandins in biopsy specimens from the antrum were not significantly different from those obtained in the body. Synthesis of both prostaglandins was significantly reduced in alcoholic patients who abstained less than five days compared with the non-alcoholic group with normal mucosa (PGE2-40%, PGF2 alpha-42% respectively). In non-alcoholic patients with severe gastritis PGE2 synthesis was increased (+30%, p < 0.05) and PGF2 alpha synthesis was decreased (-42.5%, p < 0.025). In alcoholic patients with severe gastritis PGE2 synthesis was depressed by almost 60% (p < 0.001) compared with the non-alcoholic group with severe gastritis. Neither colonisation of Helicobacter pylori nor smoking had a significant influence on the prostaglandin synthesis. CONCLUSIONS: Chronic alcohol misuse is associated with significantly reduced capacity for prostaglandin synthesis in gastric mucosa and this alcohol induced decrease in prostaglandin synthesis is modulated by the presence and degree of gastritis.     

Effect of prostaglandins E2 and F2 alpha on membrane calcium binding, Ca2+/Mg2+-ATPase activity and membrane fluidity in rat myometrial plasma membranes

Myometrial plasma membrane (MPM) preparations from rats treated with oestradiol were obtained by discontinuous sucrose-gradient centrifugation. The preparations contained calcium-stimulated and magnesium-dependent ATPase (Ca2+/Mg2+-ATPase). A dramatic decrease in the activity of Ca2+/Mg2+-ATPase was observed when preparations were treated with 0.025-10 mumol prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha)/l. In contrast, there was a marked increase in MPM-bound 5'-nucleotidase activity at low concentrations (up to 2 mumol/l) of PGE2 and PGF2 alpha; higher concentrations (up to 10 mumol/l), however, led to a progressive inhibition of enzyme activity. Association (specific and non-specific binding) of PGE2 and PGF2 alpha with MPM at pH 7 was found to require Ca2+ (half-maximal concentration approximately 0.7 mmol/l). Changes in the allosteric properties of MPM-bound 5'-nucleotidase by concanavalin A (as reflected by changes in the Hill coefficient) indicated a fluidization of the membrane induced by PGE2 and PGF2 alpha. The steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labelled MPM decreased in PGE2- and PGF2 alpha-treated MPM from 1.24 +/- 0.04 (S.D.) to 0.66 +/- 0.01 and 0.74 +/- 0.01 respectively, which is consistent with a general increase in membrane fluidity. It is suggested that PGE2 and PGF2 alpha promote changes in the physical properties of MPM which may be relevant to the induction of uterine contractions by enzymatic regulation of intracellular calcium concentrations.     

Prostaglandin E2 and F2alpha activate the FP receptor and up-regulate cyclooxygenase-2 expression via the cyclic AMP response element

In endometrial adenocarcinomas COX-2 and F-series prostanoid (FP) receptor expression and prostanoid biosynthesis (PGE(2) and PGF(2alpha)) are elevated. In the present study, we investigated the effect of PGE(2) and PGF(2alpha) on the expression of COX-2 via the FP receptor in endometrial adenocarcinoma cells stably expressing the FP receptor (FPS cells). Using chemical inhibitors of intracellular signaling pathways, reporter gene assays and quantitative RT-PCR analysis, we show that PGE(2) and PGF(2alpha) can mobilize inositol 1,4,5-trisphosphate, induce ERK1/2 phosphorylation via the phospholipase Cbeta-protein kinase A-epidermal growth factor receptor pathway and induce cyclooxygenase-2 (COX-2) expression via the FP receptor. In addition we show that the PGE(2) or PGF(2alpha)-regulation of COX-2 via the FP receptor is mediated via the cAMP response element (CRE) binding site on the COX-2 promoter. These data indicate that PGE(2) and PGF(2alpha) biosynthesized locally within endometrial adenocarcinomas can regulate tumor cell function in an autocrine/paracrine manner via the FP receptor.     

A transplantable rat Leydig cell tumour. Properties of the prostaglandin E receptor

In the present paper we have examined the properties of the prostaglandin E (PGE) receptor in a transplantable rat Leydig cell tumour (H-540). It appears that PGE1 and PGE2 share a common receptor in membrane particles from this Leydig cell tumour. From saturation analysis and modified Hofstee plots, the specific binding sites for PGE1 can be divided into a high (25%) and low affinity state (75%) with apparent equilibrium constants of dissociation (Kd) of 2.4.10(-7) mol/l and 4.4.10(-6) mol/l, respectively. Association rate kinetics at different temperatures employing 5.10(-9) mol/l [3H]PGE1 showed that specific binding was time- and temperature-dependent. At 37 degrees C an apparent steady state was achieved after approximately 4 h incubation. The binding of [3H]PGE1 was very tight and no dissociation was observed at 20 degrees C during the first 20 h. The free PGE1 receptor appears to be very unstable. Binding was reduced rapidly by storage at 0 degrees C, by freezing and thawing of membrane particles, and by incubation of concentrated membrane particles. Specificity curves showed that PGA1 and PGA2 displaced [3H]PGE1 from receptor to a somewhat lesser degree than PGE2 and PGE1, whereas PGs of the B, D, I and F series had little or no effect. The fact that inhibition of [3H]PGE1 binding by cold PGE1 occurred in the same concentration range as PGE1 activation of adenylate cyclase, indicates that the specific binding of PGE observed here represents functional receptors coupled to the adenylate cyclase.     

Molecular characterization of prostaglandin F receptor (FP) and E receptor subtype 1 (EP(1)) in zebrafish

Prostaglandins E (PGE) and F (PGF) mediate diverse physiological functions via their cell surface receptors - prostaglandin E receptor (EP) subtypes 1, 2, 3 and 4 (EP(1); EP(2); EP(3); EP(4)) and F receptor (FP). In teleost fishes, PGE was implicated in gill epithelium ion transport, while both PGE and PGF were involved in oocyte maturation, follicular rupture and coordination of reproductive behaviors. However, little is known about the mechanisms behind their actions. In present study, we first identified the full-length ORF cDNA clones of three zebrafish prostaglandin E receptor subtype 1 (zEP(1)) isoforms - zEP(1a), zEP(1b) and zEP(1c) - and FP (zFP) from adult ovary. RT-PCR showed that zEP(1a), zEP(1b) and zFP are widely expressed in adult tissues, while zEP(1c) mRNA expression is mainly confined in brain and kidney. Using a pGL3-NFAT-RE luciferase reporter system, both zEP(1a) and zEP(1b) expressed in DF-1 cells were shown to be activated by PGE(2) potently while zEP(1c) and zFP were activated by PGF(2a) effectively, suggesting that the four receptors are functionally coupled to intracellular Ca(2+)-signaling pathway. Furthermore, EP1a and EP1b, but not EP1c were suggested to couple to cAMP-PKA signaling pathway using a pGL3-CRE luciferase reporter assay. Although zEP(1c) might originate as a paralog to zEP(1a) and zEP(1b), its functional coupling to PGF(2α) instead of PGE(2) suggested that zEP(1) isoforms might have sub-functionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Characterization of these receptors undoubtedly furthered our understanding on the diverse yet highly target-specific responses of prostaglandins in teleosts.