Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.     

Effects of prostanoids on the rat's myometrium in vitro during pregnancy.

The objectives of this study were to determine the effects of pregnancy in the rat on the contractile response of the myometrium in vitro to a number of prostanoids. Longitudinally and circularly oriented strips were studied separately. Responses to PG (prostaglandin)F2 alpha, PDG2, the PGI2-mimetic iloprost, and the thromboxane (Tx) A2-mimetic U-46619 were investigated on Days 10, 15, 18, 20, 21, and 22 of pregnancy. Responses were prostanoid-dependent, and differences between longitudinal and circular strips were small. PGF2 alpha and PGD2 produced similar patterns, with a high potency but low maximal response on Day 10; thereafter potency fell to a minimum value on Day 18 and then gradually increased until Day 22, when it was still lower than at Day 10. In contrast, for PGE2 there were no changes in potency over the period of study (longitudinal muscle) or a slight increase between Days 15 and 21 (circular muscle). Both iloprost and U-46619 maintained a low potency throughout pregnancy. We conclude that the pregnant rat's myometrium is probably not a target for PGI2 or TxA2 and that the difference patterns of responses to PGE2 and PGF2 alpha during pregnancy support the hypotheses that these prostanoids act at different sites within the myometrium.     

Effects of 19-hydroxy-prostaglandins on oviductal and uterine motility.

The effects of 19-hydroxyprostaglandins (19-OH-PGs) were tested in vivo on the rabbit oviduct and uterus and on the rhesus monkey (Macaca mulatta) uterus. The 19-OH-PGEs suppressed spontaneous oviductal and uterine activity in the rabbit. The qualitative effect on the rabbit oviduct of 19-OH-PGEs was similar to that of PGE2. However, the typical response of the rabbit uterus to PGE2 was an increase in muscle activity. With regard to the rabbit oviduct, 19(R)-OH-PGE2 was as potent as PGE2, but 19(S)-OH-PGE2 was approximately 1/2 as potent as PGE2. Based on the dose of 19-OH-PGEs usually required to cause a minimal suppression and the dose of PGE2 required to cause a minimal stimulation of rabbit uterine activity, 19(R)-OH-PGE2 was twice as potent as PGE2 while 19(S)-OH-PGE2 was 1/2 as potent as PGE2. Stimulatory effects on the rabbit oviduct and uterus were observed following administration of 19-OH-PGFs and PGF2alpha. The potency on the rabbit oviduct of 19(S)-OH-PGF2alpha was about 1/5 to 1/10 that of PGF2alpha; the potency of 19(R)-OH-PGF2alpha was about 1/10 to 1/20 that of PGF2alpha. Both 19-OH-PGFs were approximately 1/5 to 1/10 as potent as PGF2alpha on the rabbit uterus. At the doses tested 19-OH-PGFs were inactive on the monkey uterus. Thus, these compounds are at least 1/5 as active as PGF2alpha. In contrast, 19(R)-OH-PGE2 had approximately the same potency as PGE2 in stimulating monkey uterine activity; but 19(S)-OH-PGE2 was approximately 1/3 as potent as PGE2.     

Effects of RU 486 on electrical activity, on sexual steroid and prostaglandin F2 alpha concentrations in the myometrium at mid-pregnancy in the rat

Effects of RU 486 (10 mg.kg-1, per.os) were assessed at mid-pregnancy in the rat. One hour after RU 486 treatment, myometrial electrical activity stayed low. It increased from the 3rd hour after administration of RU 486 and a perfect synchronization of the bursts of the action potentials was observed from the 6th h to the 24th h. Tissular steroid hormones and PGF2 alpha, evaluated at hour 6 after RU 486 administration, showed a decrease of progesterone concentrations in both myometrium and uterus. Estradiol levels decreased in uterus whereas, PGF2 alpha levels increased in both myometrium and uterus. These results show for the first time that RU 486 strongly increases the myometrial electrical activity in the rat at mid-pregnancy. This action was closely related to E2, P4 and PGF2 alpha concentrations.     

Effect of alterations in the cyclopentane ring on bone resorptive activity of prostaglandin

Previous studies have shown that the natural prostanoids, PGE2, PGE1 and PGF2 alpha are potent stimulators of bone resorption. In this study, we have examined the effects of alterations in the cyclopentane ring of these prostanoids for their effect on the resorptive response of cultured long bones from 19-day fetal rats as measured by the release of previously incorporated 45Ca. Indomethacin (10(-6)M) was added to minimize endogenous prostaglandin production. In this system PGE2 and PGE1, the 9 keto, 11 alpha hydroxy compounds, were approximately equally effective at concentrations of 10(-8) to 10(-6) M. The 9 alpha hydroxy, 11 alpha hydroxy compound, PGF2 alpha, was active at 10(-7) to 10(-5) M. In contrast, the 9 alpha hydroxy, 11-keto compound, PGD2, showed only a minimal stimulation of bone resorption at 10(-5) M. While these data suggested that the 11 alpha hydroxy group was important for bone resorbing activity, 11 beta PGE2 and 11-deoxy PGE1 were only slightly less potent than their physiologic counterparts. Both 9 beta, 11 alpha PGF2 and 9 alpha, 11 beta PGF2 were less potent than PGF2 alpha but did cause substantial stimulation of bone resorption and were equally effective at 10(-6) to 10(-5) M. 9 alpha, 11 beta PGF2 alpha is of particular interest since it is major metabolite of PGD2. These results suggest that the binding of prostanoids to the receptor which mediates bone resorption is affected by changes at the 9 and 11 positions of the pentane ring but do not support the hypothesis that the 11 alpha OH function is essential for this biological activity.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.     

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca2+ and prostaglandin E2 and F2alpha secretion from porcine myometrial cells

Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished. In conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.     

Uterine region-dependent differences in responsiveness to prostaglandins in the non-pregnant porcine myometrium

To clarify the uterine region-dependent distribution of prostanoid receptors, we compared the mechanical responses to selective prostanoid receptor agonists (FP, EP3, DP, EP2) and naturally occurring prostaglandins (PGF2alpha PGE2, PGD2) in longitudinal and circular muscles isolated from three different regions (cornu, corpus and cervix) of the non-pregnant porcine uterus. Expression levels of FP receptor and cyclooxygenase (COX-1 and COX-2) in the respective regions were also examined using RT-PCR and Western blotting. The contractile responses to fluprostenol (an FP agonist) and PGF2alpha in both longitudinal and circular muscles were strongest in the cornu but weak in the corpus and cervix. Expression levels of mRNA and protein of FP receptor were highest in the cornu, consistent with the contractile responses. ONO-AE-248 (an EP3 agonist) caused contraction of both muscle layers, but region-related difference in responsiveness was observed only in the longitudinal muscle. ONO-AE1-259 (an EP2 agonist) inhibited spontaneous contraction of the myometrium, and inhibition was conspicuously stronger in the cervix. PGE2 caused contraction (<100 nM, cornu > corpus = cervix) and inhibition (>300 nM, cornu = corpus < or = cervix) of contractility depending on the concentration in both muscle layers. BW245C (a DP agonist) inhibited the spontaneous contraction, and region-dependent different responsiveness was marked in the longitudinal muscle (cervix = corpus > cornu). COX-1 but not COX-2 was detected in the non-pregnant porcine uterus. Expression level of COX-1 was different in the longitudinal muscle (cornu > corpus = cervix) but the same in the circular muscle. SC-560 inhibited the spontaneous contraction of longitudinal muscles in all regions. The results of the present study indicate that there are region-related heterogeneous distributions of contractile (FP and EP3, cornu > cervix) and relaxant (EP2 and DP, cervix > cornu) prostanoid receptors and COX-1 in the porcine uterus. The results also suggest involvement of endogenous PGs in the regulation of spontaneous uterine contractility. Region-related differences in COX-1 and prostanoid receptors might be necessary to produce a gradient of uterine motility decreasing from the cornu to the cervix that manages movement of luminal contents.     

Profiling of bisenoic prostaglandins and thromboxane B2 in bronchoalveolar fluid from the lower respiratory tract of human subjects by combined capillary gas chromatography-mass spectrometry

Methods for the profiling of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 15(S),9 alpha,11 beta-trihydroxyprosta-5Z,13E-dien-1-oic acid (9 alpha,11 beta-PGF2), 6-keto-prostaglandin F1 alpha (6kPGF1 alpha), and thromboxane B2 (TxB2) in bronchoalveolar lavage (BAL) fluids from human subjects by combined capillary gas chromatography-mass spectrometry are described. Aliquots (5 ml) of BAL fluid obtained using a standardized lavage protocol were extracted on octadecylsilyl silica cartridges after addition of 0.8 to 2.0 nanograms of tetradeuterated analogs of PGE2, PGF2 alpha, and 6kPGF1 alpha as internal standards. Eluted analytes and internal standards were prepared for vapor phase analysis by sequential reactions resulting in the formation of methyloxime-pentafluorobenzyl ester-trimethylsilyl ether derivatives. The derivatized analytes were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The samples were of adequate purity for identification and quantitation of each of the prostanoids with detection limits of 0.1 to 0.2 picograms of each analyte per milliliter of BAL fluid. The time required for analysis of each sample was approximately 30 minutes. Standard curves of unlabeled species of the six prostanoids extracted after addition to BAL fluid were linear over a range from subpicogram to nanogram quantities. The differences between the amounts of prostanoid added and the amounts of prostanoid measured were typically less than 19%, and the intra-assay coefficients of variation for repeated measurements of a single sample were less than 20%. PGE2, PGD2, PGF2 alpha, and TxB2 were detectable in BAL fluids from normal subjects with levels of each of these compounds being less than 2.6 picograms/ml. BAL fluids from patients with lung disease presented qualitative and quantitative profiles of prostanoids markedly different than those from normal subjects. These analytical methods provide a basis for in vivo comparisons of prostanoid profiles in the lower respiratory tract of man and should be readily adaptable for use in a variety of clinical studies.     

Synthetic pathways of gallbladder mucosal prostanoids: the role of cyclooxygenase-1 and 2.

Acute cholecystitis is associated with increased gallbladder prostanoid formation and the inflammatory changes and prostanoid increases can be inhibited by nonsteroidal anti-inflammatory agents. Recent information indicates that prostanoids are produced by two cyclooxygenase (COX) enzymes, COX-1 and COX-2. The purpose of this study was to determine the COX enzymatic pathway in gallbladder mucosal cells involved in the production of prostanoids stimulated by inflammatory agents. Human gallbladder mucosal cells were isolated from cholecystectomy specimens and maintained in cell culture and studied in comparison with cells from a well differentiated gallbladder mucosal carcinoma cell line. COX enzymes were evaluated by Western immunoblotting and prostanoids were measured by ELISA. Unstimulated and stimulated cells were exposed to specific COX-1 and COX-2 inhibitors. In both normal and transformed cells constitutive COX-1 was evident and in gallbladder cancer cells lysophosphatidyl choline (LPC) induced the formation of constitutive COX-1 enzyme. While not detected in unstimulated normal mucosal cells and cancer cells, COX-2 protein was induced by both lipopolysaccharide (LPS) and LPC. Unstimulated gallbladder mucosal cells and cancer cells produced prostaglandin E2 (PGE2) and prostacyclin (6-keto prostaglandin F1alpha, 6-keto PGF1alpha) continuously. In freshly isolated normal gallbladder mucosal cells, continuously produced 6 keto PGF1alpha was inhibited by both COX-1 and COX-2 inhibitors while PGE2 levels were not affected. Both LPS and LPC stimulated PGE2 and 6 keto PGF1alpha formation were blocked by COX-2 inhibitors in freshly isolated, normal human gallbladder mucosal cells and in the gallbladder cancer cells. The prostanoid response of gallbladder cells stimulated by proinflammatory agents is inhibited by COX-2 inhibitors suggesting that these agents may be effective in treating the pain and inflammation of gallbladder disease.     

Comparison of antiprogestin stimulation of uterine prostaglandin synthesis in vitro

Progesterone has an inhibitory effect on prostaglandin synthesis in urine tissue and this effect is reversible with progesterone receptor antagonists. Although antiprogesterone steroids such as RU486 (Mifepristone) are effective at inducing abortion in women they have an improved efficacy when used with exogenous synthetic prostaglandin. In the guinea-pig such antagonists sensitize the uterus but do not result in increased myometrial activity and therefore may not induce endogenous PG synthesis. In this study the effects of antiprogestins on a preparation of rat uterus perifused with progesterone were studied. ZK98 734 caused a rapid and sustained increase in 6-oxoPGF synthesis which rose within the first 90 minutes. This rapid response suggested that some mechanism other than the induction of fresh protein synthesis was involved. A similar increase was not seen with pregnant guinea-pig myometrium/decidua perifused in a similar manner, suggesting that some other mechanism was responsible for the relatively low PG production in pregnancy. However increases in 6-oxoPGF in response to antiprogestins were recorded when pregnant guinea-pig decidua/myometrium was incubated for 4 hours. In these experiments 1 microM ZK98 734 and 1 microM ZK98 299 (Onapristone) gave a 2.7 fold increase in PG production whereas RU486 gave a 1.6 fold increase. Both 1 microM ZK98 734 and 1 microM ZK98 299 also gave a significant increase in PGE production but no increase in PGF was observed. These findings suggest that some antiprogestins might have a better effect on the stimulation of endogenous PG synthesis or on the rate of catabolism of prostanoids.     

Characterization of prostanoid receptors present on adrenergic neurons innervating the porcine uterine longitudinal muscle

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.     

Characterisation of the prostanoid receptors mediating contraction of guinea-pig isolated trachea

The receptors mediating prostanoid-induced contraction of guinea-pig isolated trachea have been characterised in terms of a recently proposed general classification of prostanoid receptors. Results obtained on the trachea were compared with those obtained on guinea-pig fundus, which contains a sub-type of PGE2-sensitive (EP-) receptor termed the EP1-receptor, and guinea-pig lung strip, which contains a thromboxane-sensitive or TP-receptor. The following agonists were studied, PGE2, PGF2 alpha and the thromboxane-like agonists U-46619 and Wy17186. The antagonists studied were SC-19220 which selectively blocks EP1-receptors, and AH19437 which selectively blocks TP-receptors. On guinea-pig fundus the rank order of agonist potency was PGE2 greater than PGF2 alpha greater than Wy-17186 approximately equal to U-46619, and responses to all agonists were antagonised by SC-19220 but not by AH19437. On guinea-pig lung strip the rank order of potency was U-46619 greater than Wy17186 much greater than PGF2 alpha greater than PGE2 and responses to all agonists tested were blocked by AH19437 but not by SC-19220. On the trachea, the rank order was PGE2 = U-46619 greater than Wy17186 = PGF2 alpha. SC-19220 antagonised responses to PGE2 and PGF2 alpha, but not those to U-46619 or Wy17186. Conversely, AH19437 antagonised responses to U-46619 and Wy17186 but not those to PGE2 or PGF2 alpha. It is concluded that prostanoid-induced contractions of guinea-pig trachea can be mediated by both EP1- and TP-receptors.     

Effects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells.

Rank order of agonist potency for activation of adenylate cyclase by the naturally occurring prostanoids PGE2, PGF2 alpha, PGD2, the stable PGI2 analogue iloprost, and the TXA2 mimetic U 46619, provides evidence for the existence of a distinct PGE-receptor on guinea-pig duodenal enterocytes. The PGE-receptor is likely to be of the EP2-subtype since the specific EP2-agonist 11-deoxy-PGE1 stimulated adenylate cyclase activity with a 20-fold higher potency than the EP1-agonist 17-phenyltrinor-PGE2 and the EP3-agonists MB 28767 and GR 63799. In addition, sulprostone (acting on both EP1- and EP3-receptors) was ineffective. Since the specific EP1-antagonist SC 19220 did not inhibit PGE2-stimulated adenylate cyclase activity, the involvement of EP1-receptors could be further excluded. The synthetic prostaglandin E-analogues misoprostol and nocloprost stimulated adenylate cyclase almost identically, though they were about 10-fold less potent than the natural PGE2.     

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.     

Prostaglandin E(2) and stem cell factor can deliver opposing signals to B lymphocyte precursors

The synthetic prostanoid, 16,16-dimethyl PGE(2), suppressed B lymphopoiesis in mice and proliferation of normal B cell precursors or the F10 pro-B cell line to interleukin 7 in culture. This was not the case with two other prostanoids, PGD(2) and PGF(2alpha), or agonists for PGI(2) agonist and thromboxane A(2) agonist receptors. PGE(2), but not the related prostanoids or agonists, induced apoptosis in F10 cells. The apoptotic response was mediated by the EP2 class of PGE(2) receptors and required an increase in intracellular cyclic adenosine 3',5'-monophosphate, activation of protein kinase A, and protein synthesis. The influence of PGE(2) on F10 cells was diminished in the presence of a cloned stromal cell line or stem cell factor. These findings describe another potential regulatory circuit in bone marrow which might influence B lymphopoiesis under disease or steady-state conditions.     

Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.     

Prostaglandin e and f receptor expression and myometrial sensitivity at labor onset in the sheep

Prostaglandins (PGs) play a pivotal role in the initiation and progression of term and preterm labor. Uterine activity is stimulated primarily by PGE(2) and PGF(2alpha) acting on prostaglandin E (EP) and prostaglandin F (FP) receptors, respectively. Activation of FP receptors strongly stimulates the myometrium, whereas stimulation of EP receptors may lead to contraction or relaxation, depending on the EP subtype (EP1-4) expression. Thus, the relative expression of FP and EP1-4 may determine the responsiveness to PGE(2) and PGF(2alpha). The aims of this study were to characterize the expression of EP1-4 and FP in intrauterine tissues and placentome, together with myometrial responsiveness to PG, following the onset of dexamethasone-induced preterm and spontaneous term labor. Receptor mRNA expression was measured using quantitative real-time polymerase chain reaction using species-specific primers. There was no increase in myometrial contractile receptor expression at labor onset, nor was there a change in sensitivity to PGE(2) and PGF(2alpha). This suggests expression of these receptors reaches maximal levels by late gestation in sheep. Placental tissue showed a marked increase in EP2 and EP3 receptor expression, the functions of which are unknown at this time. Consistent with previous reports, these results suggest that PG synthesis is the main factor in the regulation of uterine contractility at labor. This is the first study to simultaneously report PG E and F receptor expression in the key gestational tissues of the sheep using species-specific primers at induced-preterm and spontaneous labor onset.     

Prostaglandin and thromboxane synthesis by tissue slices from human aortic aneurysms

Sliced portions of the walls of human aortic aneurysms were incubated with extracts of human plasma and serum to determine the profile of prostanoid production. 6-Oxo-prostaglandin (PG) F1 alpha, PGE2, PGF2 alpha, and thromboxane (TX) B2 were measured by gas chromatography/electron capture mass spectrometry. 6-Oxo-PGF1 alpha, the stable hydrolysis product of PGI2, was the major cyclooxygenase product but substantial amounts of TXB2 (the hydrolysis product of TXA2), with smaller amounts of PGE2 and PGF2 alpha were also synthesised. These prostanoids could contribute to the response of the vascular wall to injury, thereby influencing the disease process. Serum extracts stimulated PGI2 and TXA2 synthesis, probably as a result of their Ca2+ content.