RESUMEN
We previously demonstrated that the antiprogestogen RU 486, when superfused on myometrial strips, induces a rapid decrease in spontaneous uterine contractile frequency, an increase in amplitude and duration of contractions, and a concomitant decrease in 6-keto PGF(1alpha) release. In this study, we present further work on the role of calcium transients and the involvement of the PLC/PKC pathway in mediating RU 486 effects. We found no clear causal relationship between the spontaneous contractility controlled by external Ca++ concentration and 6-keto PGF(1alpha) release depending mostly on intracellular Ca++ mobilization. We show that RU 486 strengthened the inhibitory effect of TMB8, a potent inhibitor of internal calcium, on both spontaneous contractility and 6-keto PGF(1alpha), release and antagonized the stimulatory action of thapsigargin, a toxin blocking the endoplasmic reticulum calcium pump (ER Ca++ ATPase). These data indicate that RU 486 could act as an inhibitor of intracellular Ca++ mobilization. A slight but significant decrease of the prostanoid liberation was observed in the presence of U73122, an inhibitor of PLC, but not in the presence of neomycin, another PLC inhibitory compound. PKC inhibitors, staurosporine and H7 did not significantly affect spontaneous 6-keto PGF1alpha release, showing that PIP2 hydrolysis and PKC pathway were not involved in the basal release of the prostacyclin metabolite. Vasopressin (AVP), an agent known to induce contractility of the non-pregnant human uterus, markedly increased 6-keto PGF(1alpha) release in a dose-dependent manner. Stimulation of GTP-regulated proteins (G proteins) by ALF4 was accompanied by a rise in 6-keto PGF(1alpha) liberation and a high contractile activity. The effects of both vasopressin and ALF4- were not significantly opposed by RU 486, indicating that other sources of Ca++, not controlled by the steroid, were involved in the agonist-stimulated prostanoid release. Studies with structurally related RU 486 analogues showed that the steroid effects were not dependent on their antihormonal activity, but rather on a specific 11beta arylsubstitution and a 17beta-hydroxy-13beta-methyl configuration of the 4,9-estradien-3-one molecule.
Asunto(s)
Calcio/metabolismo , Epoprostenol/metabolismo , Antagonistas de Hormonas/farmacología , Mifepristona/farmacología , Miometrio/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/metabolismo , Abortivos Esteroideos/farmacología , Arginina Vasopresina/farmacología , Bloqueadores de los Canales de Calcio/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/fisiología , Sinergismo Farmacológico , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Humanos , Líquido Intracelular/metabolismo , Mifepristona/análogos & derivados , Relajación Muscular/efectos de los fármacos , Miometrio/metabolismo , Nifedipino/farmacología , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/fisiología , Estaurosporina/farmacología , Relación Estructura-Actividad , Tapsigargina/farmacología , Contracción Uterina/efectos de los fármacos , Contracción Uterina/fisiologíaRESUMEN
We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI2 release with human myometrial strips superfused "in vitro". A decrease of PGI2 release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose-dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dose-dependent and reversible. Modification of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as 10(-9)M, dexamethasone (10(-8)M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contractility, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 oxo pregnene structure.
Asunto(s)
Epoprostenol/metabolismo , Mifepristona/farmacología , Contracción Uterina/efectos de los fármacos , Adulto , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Técnicas In Vitro , Miometrio/efectos de los fármacos , Promegestona/farmacología , Prostaglandinas F/metabolismoRESUMEN
Cytosols of rat and guinea pig liver and of human placenta were screened for their capacity to catalyze the conversion of racemic leukotriene A4 into 5S, 12R-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid (leukotriene B4). The epoxide hydrolase activities showed some specificity for the 5S,6S-oxido-(E,E,Z,Z)-7,9,11,14-eicosatetraenoic acid (LTA4) and produced mixtures of leukotriene B4 and its enantiomer containing up to 78-87% of leukotriene B4.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Leucotrieno B4/metabolismo , Leucotrienos/metabolismo , Hígado/enzimología , Placenta/enzimología , Animales , Cromatografía Líquida de Alta Presión , Citosol/enzimología , Femenino , Cobayas , Cinética , Leucotrieno A4 , Hígado/metabolismo , Placenta/metabolismo , Embarazo , Radioinmunoensayo , Ratas , Ratas Endogámicas , Especificidad por SustratoRESUMEN
It has been shown that various glutathione transferases can synthesize leukotriene C4, or its methyl ester, from glutathione and leukotriene A4. We questioned whether the same enzymes could be used to resolve racemic leukotriene A4 methyl ester (more easily prepared than the optically active enantiomer) and to produce leukotriene C4 methyl ester selectively. We present in this paper a study of the enantioselectivity of some rat liver glutathione transferase isozymes and of the glutathione transferase of human placenta for the leukotriene A4 methyl ester isomers. The rat liver 3-4 glutathione transferase exhibited the highest conversion rate but preferentially converted the (5R, 6R) leukotriene A4 methyl ester. The placental enzyme was fairly selective for the natural (5S, 6S) enantiomer but the rate of conversion was low.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Glutatión Transferasa/metabolismo , Leucotrieno A4/análogos & derivados , Leucotrieno C4/análogos & derivados , Hígado/enzimología , Placenta/enzimología , SRS-A/análogos & derivados , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Isoenzimas/metabolismo , Isomerismo , Cinética , Lipooxigenasa/metabolismo , Ratas , SRS-A/biosíntesis , Especificidad por SustratoRESUMEN
Human placental cytosol inhibits platelet aggregation induced by high doses of collagen. The aim of this study was to investigate whether this anti-aggregating activity was caused only by the presence of various activities already described in the placenta (an ADP-consuming enzyme, a fatty acid cyclooxygenase inhibitor, and a thromboxane synthetase inhibitor) or whether another factor was present. Heating the cytosol at 50 degrees C for 6 min destroyed the inhibitor of collagen-induced aggregation. ADPase and the AA pathway inhibitors were not modified by this treatment. We therefore show the presence of an additional anti-aggregating factor: it is destroyed by heating at 50 degrees C. We also tested for the presence of an inhibitor of AA release in the placental cytosol using three different methods (rabbit platelets in PRP, washed rabbit platelets, and NRK fibroblasts) but no inhibition could be evidenced. We conclude that this new anti-aggregating factor, which is probably a protein, acts neither through AA release inhibition nor AA cascade inhibition.
Asunto(s)
Placenta/fisiología , Agregación Plaquetaria , Animales , Apirasa/metabolismo , Ácido Araquidónico , Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/metabolismo , Plaquetas/metabolismo , Cromatografía en Gel , Inhibidores de la Ciclooxigenasa , Citosol/enzimología , Citosol/fisiología , Fibroblastos/metabolismo , Calor , Humanos , Agregación Plaquetaria/efectos de los fármacos , Proteínas Gestacionales/fisiología , ConejosRESUMEN
Regulation of uterine prostaglandin (PG) synthesis by steroid sex hormones was studied in female rats. Animals were ovariectomized (OVX) and received silastic implants of estradiol (E2) or progesterone (Pg); the implants were maintained for 7 days. The animals were sacrificed and their uteri homogenized at 4 degrees C. Basal levels of PGs and PGs synthesized during 20 min incubations at 37 degrees C, either without exogenous arachidonic acid (AA), or in the presence of 2.10(-5)M added AA were measured by RIA. Comparison between the various treatments shows that the regulation of uterine PG synthesis in the rat is a multistep process and depends on the type of PG. PGI2 (6 keto PGF1 alpha) is synthesised in very large amounts but is not very significantly influenced by hormonal treatment. PGF2 alpha and PGE2 are synthesized in much smaller quantities but are very dependent on hormonal treatment. E2 stimulates PGF2 alpha and inhibits PGE2, shifting the ratio from 0.5 in untreated OVX rats to 3.3 in OVX E2-treated rats. TXA2 (TXB2) is stimulated by E2. Pg significantly stimulates endogenous PGF2 alpha levels but does not change the profile of PGs synthesized from the endogenous substrate. It inhibits PGE2 synthesis from exogenously added AA. These results show that E2 favors PGF2 alpha synthesis at the expense of PGE2 and that the synthesis of PGI2, which is the main AA metabolite in the rat uterus is not hormone dependent, (at least not under the conditions of our experiments).
Asunto(s)
Ácidos Araquidónicos/metabolismo , Estradiol/farmacología , Progesterona/farmacología , Prostaglandinas/biosíntesis , Útero/metabolismo , Animales , Ácido Araquidónico , Castración , Dinoprost , Dinoprostona , Femenino , Hormonas Esteroides Gonadales/sangre , Técnicas In Vitro , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Ratas , Ratas Endogámicas , Útero/efectos de los fármacosAsunto(s)
Estradiol/farmacología , Prostaglandinas/biosíntesis , Útero/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Castración , Dinoprost , Dinoprostona , Estradiol/sangre , Estro , Femenino , Cobayas , Embarazo , Progesterona/sangre , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , Ratas , Ratas Endogámicas , Útero/efectos de los fármacosRESUMEN
Using PGH2 as substrate, we have previously demonstrated that human placenta synthesizes mainly PGE2, TxB2 and PGD2(1,2). Other reports have shown that placental tissue generates a substance which inhibits ADP-induced platelet aggregation and which was supposed to be PGI2 (3). The present study indicates that the stability of that substance is different from the stability of prostacyclin (released by umbilical artery pieces). By GC-MS and multiple ion-monitoring, we have shown the presence of 6-keto-PGF1 alpha (the stable metabolite of PGI2) in the umbilical artery incubation medium, while no trace of 6-keto-PGF1 alpha could be found in the placental medium. No conversion of AA to 6-keto-PGF1 alpha by placental microsomes was observed, even in the presence of antioxidants. The placenta possesses, in addition to the known 15-OH-PGDH and delta-13 reductase activities, a weak 9 OH PGDH which is specific for PGF2 alpha (and not PGI2 nor 6-keto-PGF1 alpha). GC-MS analysis showed that the expected metabolites of PGI2 through those three enzymes were not found in the placental medium, indicating that neither PGI2 synthesis nor metabolism could be demonstrated in the placenta.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Epoprostenol/metabolismo , Oxidorreductasas Intramoleculares , Placenta/metabolismo , Agregación Plaquetaria , Prostaglandinas/metabolismo , Arterias Umbilicales/metabolismo , 15-Oxoprostaglandina 13-Reductasa/metabolismo , 6-Cetoprostaglandina F1 alfa/metabolismo , Animales , Antioxidantes/farmacología , Dinoprost , Epoprostenol/biosíntesis , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Embarazo , Prostaglandinas F/metabolismo , RatasRESUMEN
In vitro prostaglandin biosynthesis by uteri of ovariectomized rats and guinea pigs treated or untreated with oestradiol 17 beta, administered subcutaneously, was measured by R.I.A. of PGF2 alpha and PGE2. Incubations with [1-14C] arachidonic acid were also performed and labelled metabolites were analyzed by TLC. The main metabolite in rats was 6 keto PGF1 alpha and in decreasing order of magnitude, PGF2 alpha and PGE2. In guinea pig PGF2 alpha was the main product. Ovariectomy in rats completely changed the pattern of synthetized prostanoids : PGI2 production was doubled when compared to cycling rats and PGE2 increased 10 fold. PGF2 alpha values were similar to the mean value measured during the cycle. OE2 treatment almost completely inhibited PGI2 synthesis and reduced PGE2 by half. Total PG synthesis in OE2 treated animals was decreased by 5 fold when compared to spayed rats. Endogenous PGF2 alpha synthesis was slightly stimulated. In the guinea pig OE2 treatment of ovariectomized animals increased the total synthesis from 50 per cent. PGF2 alpha was always the main metabolite. In conclusion OE2 regulation of uterine PG synthesis is depending on the animal species and cannot be explained by a unique effect on the cyclooxygenase, but rather by an interplay on the various enzymes of the arachidonic acid cascade.
Asunto(s)
Castración , Estradiol/farmacología , Prostaglandinas/biosíntesis , Útero/metabolismo , Animales , Ácido Araquidónico , Ácidos Araquidónicos/metabolismo , Dinoprost , Femenino , Cobayas , Prostaglandinas F/biosíntesis , Ratas , Ratas Endogámicas , Útero/efectos de los fármacosRESUMEN
Prostaglandin biosynthesis was studied in the rat uterus during the oestrous cycle. Uterine homogenates were incubated for 20 minutes inthe presence of exogenous substrate (2.10(-5)M). PGF2 alpha and PGE2 were measured by R.I.A.. A sharp peak of PGF2 alpha and a smaller peak of PGE2 were observed at prooestrus, 20 h. Another small PGE2 peak occurred at dioestrus II, 15 h. The lowest values of both PGs were found on dioestrus, 15 h. Plasma oestradiol concentrations were highest at proestrus, 15 h and 20 h. A sharp progesterone peak occurred at prooestrus, 20 h. The PGF2 alpha peak is next to the oestradiol peak and is superimposable or lags slightly beyond the progesterone peak. Incubation with 14C arachidonic acid and subsequent analysis of extracts by TLC and scanning showed that the major metabolite is PGI2, identified as 6 keto PGF1 alpha. The conversion rate of arachidonic acid into 6 keto PGF1 alpha is 5 times higher than into PGF2 alpha. 6 Keto PGF1 alpha was further identified by GC/MS. No significant difference was observed between 6 keto PGF1 alpha production during oestrus and dioestrus.
Asunto(s)
6-Cetoprostaglandina F1 alfa/sangre , Estradiol/sangre , Estro , Progesterona/sangre , Prostaglandinas/biosíntesis , Útero/metabolismo , Animales , Dinoprost , Dinoprostona , Epoprostenol/sangre , Femenino , Cromatografía de Gases y Espectrometría de Masas , Embarazo , Prostaglandinas E/biosíntesis , Prostaglandinas F/biosíntesis , RatasAsunto(s)
Amnios/metabolismo , Ácidos Araquidónicos/metabolismo , Oxidorreductasas Intramoleculares , Placenta/metabolismo , Endoperóxidos de Prostaglandinas Sintéticos/metabolismo , Endoperóxidos de Prostaglandina/metabolismo , Prostaglandinas H/metabolismo , Ácido Araquidónico , Dinoprostona , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Imidazoles/farmacología , Isomerasas/metabolismo , Agregación Plaquetaria , Embarazo , Prostaglandina H2 , Prostaglandina-E Sintasas , Prostaglandinas E/metabolismo , Tromboxano B2/metabolismo , Tromboxano-A Sintasa/metabolismoRESUMEN
The conversion of exogenous arachidonic acid into prostaglandins was studied in human placenta and fetal membrane microsomes. Only one prostaglandin was formed, prostaglandin E2 (PGE2), in fetal membrane microsomes. In placental microsomes PGE2 was further transformed into 15 keto-PGE2. Cofactor requirements and some characteristics of the system were studied. 1 to 3% conversion of arachidonic acid into prostaglandins was observed in placental microsomes and 5 to 8% conversion in fetal membrane microsomes.