Prostaglandin E2 Alpha Research Articles

Tumor risk biomarkers and physical activity in type 2 diabetes, patients with colorectal cancer and individuals without diabetes

Biomarkers such as 8-oxo-2’deoxy-guanosine (8-oxo-dG), prostaglandin E2 alpha (PGE2 alpha), prostaglandin metabolites (PGEM) and 8-isoprostane (8-iso) may quantitatively reflect individual tumor risk including colorectal cancer (CRC) and may be useful in clinical management and prevention programs. To further clarify their role in a clinical setting we compared urinary excretion levels with regard to physical activity and other clinical and metabolic parameters in patients with longstanding (>5 years) type 2 diabetes (T2D) (moderate risk) and in those with curatively treated CRC (high risk for recurrence). Patients and methodsIn this one center case-control study 60 patients with T2D and 40 patients with CRC were compared with 40 healthy controls. Levels of physical activity were assessed in MET-hr/wk. Urinary excretion of 8-oxo-dG, PGE2 alpha, PGEM and 8-iso were determined by HPLC-ESI mass-spectrometry and ELISA, respectively. ResultsUrinary 8-oxo-dG excretion was 33.7% higher in T2D compared to controls (P=0.01), in CRC excretion was 83.6% higher (p<0.0001). There was a positive correlation with BMI (p=0.04) in all study participants. There were no significant correlations with the levels of physical activity and other clinical and metabolic parameters (HbA1c, pre-, post-prandial glucose). Urinary excretion of PGE2alpha, PGEM and 8-iso were similar in all study participants. ConclusionsThis study provides quantitative biomarker data which may be useful in management and prevention programs. One important clinical application would be the implementation of biomarker data into risk prediction models to improve accuracy. The lack of correlation with the levels of physical activity does not preclude their value in interventional settings.

Indomethacin inhalation therapy for patients with excessive sputum

We studied the effect of indomethacin (INDO) inhalation therapy on patients with excessive sputum due to chronic respiratory infection. INDO (3.5 x 10(-2) M) reduced sputum production from 211 +/- 43 to 106 +/- 22 ml/day (p less than 0.05, n = 7), the effect that was more remarkable in patients with diffuse panbronchiolitis (DPB). The prostaglandin E2 (PGE2) levels in sputum were also decreased significantly. In addition, to elucidate the mechanism of action of INDO on epithelial ion transport, we measured short-circuit current (Isc) of canine tracheal epithelium in response to 3 x 10(-6) M INDO under short-circuit conditions in vitro. Addition of INDO to mucosal medium decreased Isc from 42.8 +/- 6.0 to 8.2 +/- 3.5 microA/cm2 (p less than 0.01, n = 6). The increases in Isc produced by PGE2 and PGF2 alpha were abolished by furosemide, a Cl-transport blocker. These results suggest that INDO inhalation is a promising therapy in improving excessive sputum production, and that this effect may be attributed to inhibition of PGE2-dependent Cl-secretion into the airway lumen.

Expression of myometrial activation and stimulation genes in a mouse model of preterm labor: myometrial activation, stimulation, and preterm labor.

Myometrial contractions of labor result from an increase in myometrial activation and stimulation. Activation develops through the expression of contraction associated proteins (CAPs), including oxytocin receptors (OTR), connexin-43 (Cx-43), and prostaglandin F2 alpha, receptors (FP). Stimulation involves increases in contractile agonists including prostaglandin E2 (PGE2) and prostaglandin F2 alpha. (PGF2 alpha) that may result from increases in prostaglandin endoperoxide H synthase (PGHS)-2. A mouse model of preterm birth was used to study gene expression involved in myometrial activation and stimulation. To induce preterm birth, pregnant C57BL/6J mice were intubated with 6 g/kg ethanol on gestational day 16 and were killed every 6 h from treatment until birth. RIA was used to measure uterine PGE2 and PGF2 alpha, while PGHS-2, OTR, Cx-43, and FP messenger RNA levels were measured by ribonuclease protection assay. Increases in CAP mRNA were associated with term and preterm birth. There were differences in stimulation effectors associated with preterm and term birth. Uterine PGF2 alpha values were increased only at the time of term birth, but PGE2 was elevated during both preterm and term labor. These data suggest that existing levels of PGF2 alpha are sufficient for preterm birth when CAP expression is increased, but term labor requires increases in PGE2, PGF2alpha, and CAPs. The PGHS-2 messenger RNA expression pattern suggests that it is a CAP.

In vivo administration of nimesulide, a selective PGHS-2 inhibitor, increases in vitro myometrial sensitivity to prostaglandins while lowering sensitivity to oxytocin.

To investigate the action of prostaglandin synthesis inhibitors on uterine sensitivity to established stimulants of myometrial activity, we tested the effect of in vivo administration of nimesulide, a selective cyclooxygenase-2 inhibitor, into ovariectomized nonpregnant sheep on subsequent in vitro myometrial responsiveness to prostaglandin E2 (PGE2), PGE2 alpha, and oxytocin. Sixteen ovariectomized ewes were infused intravenously with either estradiol (2 micrograms h, n = 8) or estradiol plus nimesulide (20 mg h, n = 8); for 48 h before necropsy. Longitudinal strips of myometrium were superfused with Krebs buffer solution and their spontaneous baseline contractility recorded after 1 hour. pD2 values and maximum percentage increases in contractile tension were derived from dose-response curves to 10(-9) to 10(-5) mol/L PGE2 and PGE2 alpha, and 10(-11) to 10(-7) mol/L oxytocin. Baseline activity was lower in myometrium from estradiol-treated ewes infused with nimesulide as compared with ewes infused with estradiol alone. Maximum tension increase in response to all three agonists was similar in myometrium from estradiol- and nimesulide-treated ewes, but the pD2 in myometrium from nimesulide-treated ewes was higher in response to PGE2 and PGE2 alpha and lower in response to oxytocin. The results show that nimesulide lowers spontaneous myometrial contractility and sensitivity to oxytocin while increasing sensitivity to PGs, indicating a down-regulation of oxytocin receptor and an up-regulation of PG receptors and/or intracellular signaling events.

Involvement of prostaglandin E2 and interleukin-1 alpha in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-1 alpha (IL-1 alpha) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-1 alpha and prostaglandin E2 (PGE2) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-1 alpha and PGE2 are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-1 alpha antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE2 and IL-1 alpha synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.

Inflammatory mediators of the terminal dentition in adult and early onset periodontitis.

Based upon the prosthodontic literature, subjects who are at the transition stage between natural dentition and edentulism are called "terminal dentition" (TD) cases. The aim of the present cross-sectional investigation was to characterize the local and systemic inflammatory responses in 2 groups of patients with terminal dentition periodontitis. Eight severe adult periodontitis terminal dentition (AP-TD) subjects and 8 early onset periodontitis terminal dentition (EOP-TD) subjects were entered into the study. Our purpose was to measure an extended battery of cytokines in the gingival crevicular fluid (GCF) and in lipopolysaccharide (LPS)-stimulated monocytic culture supernatants as well as gingival mononuclear cell messenger RNA (mRNA) transcripts determined from biopsy samples. Within the GCF there were 3 tiers (levels) of mediators based upon approximate 10-fold differences in concentration. The highest tier included prostaglandin E2 (PGE2), interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2), the intermediate tier included tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN-gamma) and at the lowest concentration level were interleukin-4 (IL-4) and interleukin-6 (IL-6). Thus, the GCF analysis clearly indicated that in both AP-TD and EOP-TD groups the monocytic, i.e. IL-1 beta and PGE2 and Th1, i.e. IL-2 and IFN-gamma, inflammatory mediator levels quantitatively dominated over the Th2 mediators, i.e. IL-4 and IL-6. LPS-stimulated monocytic release of IL-1 beta, PGE2 and TNF alpha was significantly elevated in both AP-TD and EOP-TD groups compared to those of a control group of 21 subjects with moderate to advanced adult periodontitis. The cytokine mRNA expression of isolated gingival mononuclear cells showed that in both the AP-TD and the EOP-TD groups Th1 and Th2 cytokines were expressed, with low levels of IL-4 and IL-12. In conclusion, our data suggest that this cross-sectional TD periodontitis model may reflect progressive periodontal disease associated with tooth loss. Furthermore, although Th1 cytokine levels in the GCF dominate over the Th2 response, monocytic activation provides the main source of proinflammatory mediators. In addition, LPS-stimulated peripheral blood monocytes demonstrate an upregulated inflammatory mediator secretion in the terminal dentition.

Evidence that hormone receptors couple and activate a common signal transducer adenylyl cyclase in corpus luteum

To investigate whether membrane-bound hormone receptors in corpus luteum (CL) couple to one common adenylyl cyclase (AC) or whether each receptor is coupled to its own AC. Plasma membranes from rat CL were used to assess the coupling of hormone receptors to AC under conditions allowing full expression of the enzyme system. The response to hCG, prostaglandin E2 alpha (PGE2), and isoproterenol were analyzed. Adenylyl cyclase activity was monitored by the direct conversion of [infinity-32P]ATP into [32P]cyclic AMP. Results were expressed as pmol/min per mg membrane protein. Addition of hCG (200 mIU), PGE2 (100 microM), and isoproterenol(100 microM) in the presence of a saturating (100 microM) concentration of guanine nucleotide resulted in a marked stimulation of the enzyme compared with controls, reaching 552 +/- 28, 537 +/- 42, and 558 +/- 32 (mean +/- SEM), respectively. Addition of hormones in pairs or all three together did not result in an additive response of the individual effects. Thus, in the presence of the three hormones together, AC stimulation was 582 +/- 41 (n = 5), a value that was not significantly different from the stimulation observed with each hormone alone. These data indicate that luteal membranes contain a single AC system. Therefore, hormone receptors couple and activate a common signal transducer AC in luteal membranes.

Influence of sex steroids on the production of prostaglandins F2 alpha and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium.

The uterus is a primary target for sex steroid action in vivo during the estrous cycle and pregnancy. Cell cultures have been used to determine the specific function of the different cell types forming the uterus. We used endometrial cell cultures previously characterized in our laboratory to study the effect of estradiol (E) and progesterone (P4) on prostaglandin (PG) production and on regulation of the response of the cells to oxytocin (OT). The studies were performed on confluent cultures of epithelial cells grown as a monolayer either on plastic or on filter inserts to allow basal-apical polarization. As described previously, prostaglandin F2 alpha (PGF 2 alpha) production was greater (3.7-fold, p < 0.0001) than prostaglandin E2 (PGE2) production in epithelial cells, and the opposite was true in stromal cells (PGE2 9.9-fold > PGF2 alpha, p < 0.0001). In epithelial cells, the basal production of PGE2 (-61.6%, p < 0.0001) and PGF2 alpha (-51.7%, p < 0.0001) was reduced significantly by E and increased significantly by P4 (PGE2, + 30.0% [p < 0.002]; PGF2 alpha, + 22.2% [p < 0.006]). No significant effect of sex steroids on the basal production of PGs was detected in stromal cells. OT stimulated the production of PGF2 alpha (6.7-fold, p < 0.0001) and PGE2 (9.1-fold, p < 0.0001) in epithelial but not stromal cells. Treatment of the cells with E significantly (p < 0.001) increased OT-stimulated PGF2 alpha production in both the epithelial and stromal cells and that of PGE2 in epithelial cells only. The effect of steroids and OT was similar in polarized (filter) and nonpolarized (plastic) epithelial cells. Analysis of the vectorial secretion of PGs in epithelial cells grown on filter inserts revealed that PGF2 alpha is preferentially secreted in the basal (p < 0.001) compared to the apical compartment. The direction of secretion was not influenced by steroid or OT treatments. The results suggest that epithelial cells of the endometrium are a preferred target for the regulation of PG synthesis by sex steroids and OT.

Effects of prostaglandin E2 and F2α on the skeleton of osteopenic ovariectomized rats

This article contains the histomorphometric evaluation of the effects of prostaglandin F2 alpha (PGF2 alpha) on cancellous bone from the lumbar vertebra and cortical bone from the tibial shaft of ovariectomized, osteopenic rats. These effects were then compared with those of prostaglandin E2 (PGE2). Three-month-old rats were either ovariectomized (ovx) or sham-ovx. Then, either PGF2 alpha or PGE2 in doses of 1 and 3 mg/kg/day was given subcutaneously for 21 days at 150 days post ovx. Histomorphometric analysis was performed separately on both the primary and secondary spongiosae of the fourth lumbar vertebral bodies (LVB) and on tibial shafts. The ovx rats exhibited osteopenia in both primary (-23% to -37%) and secondary (-20%) spongiosae of the LVB, but not in the tibial shafts at 150 and 171 days post ovx. In the LVB, PGE2 in doses of 1 or 3 mg/kg/day for 21 days restored trabecular bone volume to the levels of sham-ovx controls in the primary spongiosa. However, in the secondary spongiosa, the treatments only thickened the trabeculae. The effects of the PGF2 alpha treatment were similar to those of the PGE2 in both the primary and the secondary spongiosae. While both PGF2 alpha and PGE2 treatments stimulated bone formation in the LVB as indicated by the increases in labeled perimeter, tissue and bone area-based bone formation rates, PGE2 is about 10 times more potent than PGF2 alpha in these effects. The PGE2 treatment also elevated activation frequency in the LVB, while the PGF2 alpha treatment did not. The treatments differed in that PGE2 at these dose levels did not alter the eroded surface in the LVB while PGF2 alpha decreased it significantly. Thus, the increase of the ratio of labeled to eroded perimeter in the LVB in PGF2 alpha-treated animals was much more than that in PGE2-treated animals. In the tibial shafts, PGE2 in doses of 1 and 3 mg/kg/day produced new marrow trabeculae in 2 of 6 and 3 of 6 of the ovx rats. However, no new trabecula was found in PGF2 alpha-treated tibial shafts. Higher doses of PGE2 also increased periosteal labeled perimeter, MAR, and BFR/BS, while PGF2 alpha did not produce any significant change in these parameters. Both PGE2 and PGF2 alpha in doses of 1 and 3 mg/kg/day increased the labeled perimeter, MAR and BFR/BS and decreased the eroded perimeter in the endocortical surface. We concluded that both PGF2 alpha and PGE2 in doses of 1 and 3 mg/kg/day for 21 days exhibited anabolic bone effects. The effects were mostly confined to an increase in trabecular volume in the primary spongiosa of the LVB and in the endocortical surface of tibial shafts. The tissue level mechanism behind this appears to be that PGE2 and PGF2 alpha can both stimulate osteoblast recruitment and activity. Overall, we found PGE2 to be more potent than PGF2 alpha at the same dose level at the endocortical surface. Furthermore, new marrow trabecular bone formed only after PGE2 treatment. PGF2 alpha differed from PGE2 by significantly reducing the trabecular eroded surface in ovx rats.

Arachidonic acid metabolites and glucocorticoid regulatory mechanism in cultured porcine tracheal smooth muscle cells.

To elucidate the signal transduction system in the production of prostaglandin E2 (PGE2) by porcine tracheal smooth muscle cells in culture (PTSMC), we examined the pattern of arachidonic acid metabolites released from PTSMC and the relationship between bradykinin-stimulated rises in intracellular calcium concentration ([Ca2+]i) and PGE2 production by PTSMC. We next examined the effect of dexamethasone on these parameters. Bradykinin induced a dose-dependent increase in both the rise in [Ca2+]i and PGE2 production by PTSMC. The increase in [Ca2+]i paralleled an increase in PGE2 production. High-performance liquid chromatography (HPLC) revealed that dexamethasone-treated PTSMC were suppressed to release arachidonic acid metabolites such as PGE2 and prostaglandin F2 alpha (PGF2 alpha). Incubation of PTSMC with 10(-6)M dexamethasone for 24 h significantly suppressed both the rise in [Ca2+]i and PGE2 production by PTSMC in response to bradykinin, and also significantly suppressed bradykinin-stimulated release of radioactivity from PTSMC prelabeled with 3H-labeled arachidonic acid (3H-AA). When PTSMC pretreated with dexamethasone were incubated with 170 nM prostaglandin H2 (PGH2) or 20 microM arachidonic acid; PTSMC synthesized less PGE2 than control PTSMC. Results suggest that bradykinin stimulates PTSMC to produce PGE2 via the signal transduction system including Ca2+, and dexamethasone appeared to suppress PGE2 production by reducing the activity of cytosolic phospholipase A2 (cPLA2) and PGE2 synthase. However, we failed to demonstrate the suppression of the activity of cyclooxygenase in PTSMC by dexamethasone. Since the elevation of [Ca2+]i is necessary for the contraction of airway smooth muscles, dexamethasone seems to reduce the contraction of airway smooth muscles by suppressing the rise in [Ca2+]i and the release of arachidonic acid metabolites. Reduced production of arachidonic acid metabolites may also contribute to improvement in the bronchial inflammation.

Amphibian oocyte: a model of a possible regulatory mechanism for prostaglandin E2 and prostaglandin F2 alpha synthesis.

To clarify the possible mechanisms regulating prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) synthesis, the effects of gonadotropin-releasing hormone (GnRH) and substance P (SP) on the release of these two prostaglandins were studied in the oocytes of the crested newt, Triturus carnifex. Full-grown oocytes of T. carnifex, freed from follicular cells, were incubated in the presence of GnRH or SP and of the inhibitors of several enzymes involved in the release of arachidonic acid (AA) and in the conversion of AA into PGE2 and PGF2 alpha. In parallel, the same experiments were performed on oocytes with membrane phospholipids labelled with [3H]AA. In addition, the PGE2-9-ketoreductase activity was evaluated through the conversion of [3H]PGE2 into [3H]PGF2 alpha. The results showed that GnRH and SP could regulate prostaglandin synthesis through the activation of phospholipase C and diacylglycerol lipase, and through the modulation of PGE2-9-ketoreductase in the oocytes of T. carnifex. In particular, GnRH enhances the activity of PGE2-9-ketoreductase with a consequent increase in PGF2 alpha, while SP inhibits the enzyme which leads to an increase in PGE2. A similar mechanism could also be hypothesized for other vertebrate species.

Arachidonic acid and its metabolites effects on testosterone production by rat Leydig cells

Arachidonic acid (AA) seems to play an important role in testicular steroidogenesis, although controversial data exist in the literature. In the present study AA induced a dose related increase of testosterone (T) formation and, at the highest dose, stimulated the production of prostaglandin E2 (PGE2), leukotrienes B4 (LTB4) and C4 (LTC4) by purified rat Leydig cells. The contemporary addition of the prostaglandin synthesis blocker, indomethacin (IND), and AA further increased T formation, decreased PGE2 levels and did not modify LTB4 and LTC4 concentrations. The addition of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA, 5 microM), did not influence the stimulatory effect of AA on T and PGE2 formation while it decreased the output of LTB4 and LTC4. When 20 microM NDGA was used in addition to AA the expected reduction of leukotrienes release was observed together with a surprising impairment of T and PGE2 secretion. PGE2 and PGF2 alpha did not modify basal T production but reduced HCG-stimulated T secretion at the 10 nM dose. When 5-12- and 15-HETE were tested an enhancement of basal T formation was observed at the 10nM dose. 5-HETE (10nM) stimulated HCG-induced T production. LTA4, LTB4 and LTE4 did not influence basal T output while LTC4 and LTD4 inhibited it. LTC4 (10nM) induced a decrease of HCG-stimulated T production. These findings suggest that: 1) exogenous AA stimulates T secretion; 2) conversion of AA to cycloxygenated and lipoxygenated metabolites is not required for its steroidogenic effect; 3) cycloxygenated and lipoxygenated compounds play a diverse modulatory role on testicular steroidogenesis.

Differential cyclooxygenase-1 and -2 gene expression in human myometria from preterm and term deliveries.

The expression of cyclooxygenase-1 (COX-1) and COX-2 genes in myometria from preterm and term pregnancies was investigated by in situ hybridization, immunoblotting, and immunocytochemistry. In situ hybridization revealed that myometrial smooth muscle contained both COX-1 and COX-2 messenger ribonucleic acid transcripts. Immunoblotting revealed that myometria contained 55- and 67-kilodalton COX-1 proteins as well as 72- and 56-kilodalton COX-2 proteins. Immunocytochemistry showed that COX-1 and COX-2 proteins are present in the myometrial smooth muscle. The expression of COX-1 and COX-2 genes differed in myometria. For example, although COX-1 expression was lower, COX-2 expression was higher at term compared to preterm pregnancy not in labor. In addition, although COX-1 expression was not dependent on labor, COX-2 expression was lower with labor compared to not in labor for both preterm and term pregnancy. The myometrial smooth muscle also immunostained for prostaglandin E2 (PGE2) and PGF2 alpha, suggesting that the cyclooxygenases are catalytically active. The changes in PGE2 and PGF2 alpha paralleled the changes in COX-1 enzyme in terms of being lower at term compared to preterm pregnancy and the changes in COX-2 enzyme in terms of being lower during labor at preterm and term pregnancy. In summary, our results demonstrate that pregnant human myometria express both COX-1 and COX-2 genes. The expression of these genes differed in preterm or term pregnancy myometria from in labor and not in labor.

Ontogenic increase in PGE2 and PGF2α receptor density in brain microvessels of pigs

1. The hypothesis that the relative vasoconstrictor ineffectiveness of prostaglandin E2 (PGE2) and PGF2 alpha on cerebral vessels of newborn pigs might be due to fewer receptors for these prostanoids was tested by comparing receptors for PGE2 (EP) and PGF2 alpha (FP) in cerebral microvessels from newborn and adult pigs. 2. Specific binding of [3H]-PGE2 and [3H]-PGF2 alpha to membranes prepared from brain microvessels showed that EP and FP receptor density (Bmax) in tissues from newborn animals was less than 50% of that determined in tissues from adults. By contrast, estimates of affinity (KD) were unchanged. 3. Specifically bound [3H]-PGE2 to brain microvessels from both the newborn and adult was displaced by AH 6809 (EP1-selective antagonist) by 80-90%, and only by approximately 30-35% by both 11-deoxy PGE1 (EP2/EP3 agonist) and M&B 28,767 (EP3 agonist); butaprost (EP2 agonist) was completely ineffective. 4. PGE2, 17-phenyl trinor PGE2 (EP1 agonist), PGF2 alpha and fenprostalene (PGF2 alpha analogue) caused significantly less increase in inositol 1,4,5-triphosphate (IP3) in brain microvessels from the newborn than in those from adult pigs. The stimulation of IP3 by PGE2 and 17-phenyl trinor PGE2 was almost completely inhibited by the EP1 antagonist, AH 6809. 5. PGE2, 11-deoxy PGE1 and M&B 28,767 produced small reduction of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in adult vessels but no effect in newborn tissues. 6. The lower density of EP and FP receptors in microvessels of newborn pigs compared to adults may explain the reduced ability of PGE2 and PGF2 alpha to stimulate production of IP3 in tissues from newborn animals. This in turn, may provide an explanation for previous observations demonstrating that these prostanoids elicit contraction of adult cerebral microvessels, but exert minimal effects on these vessels in newborn animals.

Role of nitric oxide in eicosanoid synthesis and uterine motility in estrogen-treated rat uteri.

Cholinergic stimulation of vascular endothelin activates NO synthase (NOS), leading to generation of NO from arginine. This NO diffuses to the overlying vascular smooth muscle and causes vasodilatation. NOS has also been found in the central and peripheral nervous systems and it is clear now that NO plays an important role as a neurotransmitter. Here we investigate the role of NO in controlling contraction of uterine smooth muscle. Our previous work showed that NO activates the cyclooxygenase enzyme in the hypothalamus, leading to production of prostaglandin E2 (PGE2). We began by determining whether NO was involved in production of arachidonic acid metabolites in the uterus. Uteri were removed from female rats that had been treated with estrogen (17 beta-estradiol). Control animals were similarly injected with diluent. Tissues were incubated in vitro in the presence of [14C]arachidonic acid for 60 min. Synthesis of PGs and thromboxane B2 (TXB2) was markedly stimulated by sodium nitroprusside (NP), the releaser of NO. The effect was greatest on TXB2; there were no significant differences in increases of different PGs. The response to NP was completely prevented by Hb, a scavenger of NO. The inhibitor of NOS, NG-monomethyl-L-arginine (NMMA), significantly decreased synthesis of PGE2 but not the other prostanoids (6-keto-PGF1 alpha and PGF2 alpha). Addition of Hb to scavenge the spontaneously released NO inhibited synthesis of 6-keto-PGF1 alpha, PGE2, and PGF2 alpha, but not TXB2. There was a much lesser effect on products of lipoxygenase, such that only 5-hydroxy-5,8,11,14-eicosatetraenoic acid (5-HETE) synthesis was increased by NP, an effect that was blocked by Hb; there was no effect of NMMA or Hb on basal production of 5-HETE. Thus, NO stimulates release of the various prostanoids and 5-HETE; blockade of NOS blocked only PGE2 release, whereas Hb to scavenge the NO released also blocked synthesis of 6-keto-PFG1 alpha, PGE2, and PGF2 alpha, indicating that basal NO release is involved in synthesis of all these PGs, especially PGE2. Presumably, NMMA did not block NOS completely, whereas Hb completely removed released NO. This may explain the different responses of the various prostanoids to NMMA and Hb. To determine the role of these prostanoids and NO in control of spontaneous in vitro uterine contractility in the estrogen-treated uterus, the effect of blocking NOS with NMMA and of scavenging NO produced by Hb on the time course of spontaneous uterine contractility was studied. Surprisingly, blockade of NOS or removal of NO by Hb prevented the spontaneous decline in uterine motility that occurs over 40 min of incubation. We interpret this to mean that NO was released in the preparation and activated guanylate cyclase in the smooth muscle, resulting in production of cGMP, which reduces motility and induces relaxation. When the motility had declined to minimal levels, the effect of increased NO provided by NP was evaluated; apparently by stimulating the release of prostanoids, a rapid increase in motility that persisted for 10 min was produced. This effect was completely blocked by Hb. The action of NO was also blocked by indomethacin, indicating that it was acting via release of PGs. Apparently, when motility is low, activation of PG synthesis by NO to activate the cyclooxygenase enzyme causes a rapid induction of contraction, whereas, when motility is declining, NO acts primarily via guanylate cyclase to activate cGMP release; the action of the prostanoids released at this time is in some manner blocked.

Induction of cyclooxygenase and prostaglandin E2 production by the bovine pre-embryo

A study was conducted to determine the induction of cyclooxygenase production in the cleaved and non-cleaved oocyte and to measure the secretion of prostaglandin E2 (PGE2), PGF2 alpha and tumour necrosis factor-alpha (TNF alpha) in the conditioned medium of non-mature oocytes (denuded), mature oocytes (oocyte-cumulus complex), cleaved oocytes, and non-cleaved oocytes (mature oocytes incubated in the absence of sperm). Cyclooxygenase was readily detectable in 48-h cleaved oocytes whereas the signal for the enzyme in the non-cleaved oocytes was weak or undetectable. Cyclooxygenase expression in the cleaved oocytes was transient and enzyme concentrations at the early morula stage (72 h) were low. Measurable amounts of both PGE2 and TNF alpha were secreted during the first 24 h of maturation and 6 h after fertilization, whereas PGF2 alpha was undetectable at these times. There was a dramatic enhancement in PGE2, PGF2 alpha and TNF alpha secretion 48 h after fertilization. At 72 h after fertilization, PGE2 concentrations were much lower and PGF2 alpha was again undetectable which corresponded to a low concentration of cyclooxygenase. In contrast, high TNF alpha activity continued to be detected even 72 h after fertilization. It is possible that the induction of the production of cyclooxygenase and the secretion of PGs and TNF alpha by fertilized oocytes at specific times after fertilization plays a role in the regulation of embryonic development, maternal immunological recognition of pregnancy, and the maintenance of a suitable hormonal environment for embryonic viability.

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro.

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.

Prostaglandin E2 and prostaglandin F2 alpha involvement in the corticosterone and cortisol release by the female frog, Rana esculenta, during ovulation.

Interrenal and ovarian tissues of Rana esculenta were incubated in vitro during the preovulatory, ovulatory and postovulatory phases to study the basal release of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), corticosterone, and cortisol. The effects of exogenous PGE2 and PGF2 alpha on interrenal and ovarian corticosteroid release were also studied. In addition, the plasma values of these four hormones were assessed during the same phases. During in vitro interrenal incubations, PGE2, PGF2 alpha, corticosterone, and cortisol basal releases were higher in the postovulatory phase, PGE2 and PGF2 alpha treatment in vitro increased corticosteroids during the ovulatory phase. During in vitro ovarian incubations, PGE2 basal release was higher in the preovulatory phase and PGF2 alpha and corticosteroids in the ovulatory phase; PGE2 treatment in vitro decreased corticosteroids in the ovulatory phase, and PGF2 alpha increased corticosteroids in the preovulatory and postovulatory phases. PGE2, corticosterone and cortisol plasma values were higher during the postovulatory phase, while PGF2 alpha was elevated during the ovulatory phase. These findings suggest that ovarian corticosteroids could be considered one of the factors inducing ovulation and that their synthesis may be modified by PGs.

In vitro production of cytokines by bone surface-derived osteoblastic cells in normal and osteoporotic postmenopausal women: relationship with cell proliferation.

To evaluate the role of cytokines produced by osteoblasts in the pathophysiology of bone lesions in postmenopausal osteoporosis (PMOP), we have determined by RIA and immunoradiometric assays the levels of prostaglandin E2 (PGE2), interleukin-1 beta (IL-1), tumor necrosis factor-alpha (TNF alpha), and IL-6 released by cultured bone surface-derived osteoblastic (OB) cells isolated from 24 untreated PMOP women with high, low, or normal bone turnover on bone biopsy. OB cells isolated from patients with high bone formation had a 2-fold increased proliferation rate in vitro compared to OB cells from patients with normal or low bone formation or OB cells from age-matched controls. The spontaneous in vitro production per cell protein of PGE2, IL-1, and TNF alpha, but not of IL-6, was 2- to 3-fold lower in rapidly proliferating OB cells isolated from PMOP patients with high bone formation compared to OB cells from patients with normal or low proliferation or control cells. Treatment with 10 nmol/L 1,25-dihydroxyvitamin D (48 h) increased PGE2 levels to normal values in OB cells with a high proliferation rate, but decreased PGE2 production in cells with low proliferation and in control cells, suggesting that the release of PGE2 was dependent on the stage of maturation of OB cells. Significant correlations were found between IL-1 and TNF alpha (r = 0.87; P < 0.001), IL-1 and PGE2 (r = 0.46; P < 0.05), IL-6 and IL-1 (r = 0.39; P < 0.05), and IL-6 and TNF alpha (r = 0.49; P < 0.05), suggesting that the production of these cytokines was under reciprocal control. The results indicate that the in vitro production of PGE2, IL-1, and TNF alpha, but not IL-6, by OB cells isolated from patients with PMOP is related to the proliferation rate of these cells and the rate of bone formation. The variable production of cytokines by OB cells may contribute to the histological heterogeneity of bone formation in PMOP.

Effects of individual and combined treatment with prostaglandins E2 and F2 alpha on progesterone secretion by ovine luteal cells supplemented with homologous serum lipoproteins in vitro.

Prostaglandin F2 alpha (PGF2 alpha) is a potent luteolysin, whereas prostaglandin E2 (PGE2) is generally luteotropic in vivo. To establish a model system for investigations of the mechanisms involved in these actions, we examined the effects of individual and combined treatment with PGE2 and PGF2 alpha on basal and ovine LH-stimulated progesterone secretion during long-term incubations conducted with and without supplemental homologous low-density lipoprotein (LDL) as substrate. Effects of both PGF2 and PGF2 alpha were concentration- and time-dependent and were further influenced by the presence of LDL and/or LH in medium. Neither of the prostaglandins exerted any significant effect before 48 h in culture, but distinctly different patterns of response to PGE2 and PGF2 alpha emerged thereafter. Low, but not high, concentrations of PGE2 increased progesterone secretion in the absence of LH, whereas PGF2 alpha (alone and in combination with PGE2) inhibited progesterone production in all medium formulations. The transcriptional inhibitor actinomycin effectively blocked the actions of PGF2 alpha, but had no effect on response to LH or PGE2. These data demonstrate that both the putative luteotropic actions of PGE2 and the potent, luteolytic effects of PGF2 alpha in vivo can be reproduced in long-term cultures of ovine luteal cells in vitro, and they suggest that the mechanism of PGF2 alpha-induced luteolysis may involve new protein synthesis.