M.F. Gimeno

Melatonin blocks in vitro generation of prostaglandin by the uterus and hypothalamus

The effects of melatonin on the motility (isometric tension developed and contractile frequency) of uterine horns isolated from ovariectomized rats as well as on the mechanical responsiveness to added oxytocin or prostaglandin F2 alpha (PGF2 alpha) were explored. The pineal indole (10(-6) M or higher) depressed significantly the spontaneous motility of the uterus and reduced the responses evoked by oxytocin but not those evoked by PGF2 alpha. Melatonin was also tested on the prostaglandin (PG) release into the suspending media from either uterine horns from spayed rats or bovine medial basal hypothalamic (MBH) explants. Melatonin (10(-3) M) diminished the output from the uterus or the MBH of both PGE and PGF-like material. Similarities between the effects of melatonin and indomethacin as well as the possible physiological relevance of the present findings are discussed.

The effect of estradiol on isolated rat uterine motility and on prostaglandin generation.

The motility of isolated uterine horns as well as the generation of PGE and PGF like material by the uterus from estrus and spayed rats, treated or untreated with 17-beta estradiol, were studied. Following 40 minutes of mounting the spontaneous motility of uteri from estrus rats had a lower magnitude than that from spayed ones. The amount of PGF-like material was similar in both groups whereas the first one liberated less PGE-like substance. In spayed animals treated wth 1 microgram of 17-beta estradiol the decay of spontaneous c ontractile force was higher that that observed in untreated rats, and similar to that displayed by uteri from estrus. Less PGE-like material was liberated in comparison with spayed animals and a tendency to produce higher quantity of PGF-like compounds was observed, although the level was not significantly different. With 50 micrograms of 17-beta estradiol the spontaneous reduction of contractile activity was higher than in spayed animals and than in those treated with 1 microgram. The amount of PGF-like material liberated was higher than in spayed rats and less PGE-like substance was generated comparing with spayed and 1 microgram-treated animals. These findings show that estradiol decreases the release of PGE-like compound. It would also appear that this may have some relationship with the levels of spontaneous contractile activity of the isolated rat uterus.

Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture

The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.

Human plasma transforms prostacyclin (PGI2) into a platelet antiaggregatory substance which contracts isolated bovine coronary arteries

Prostacyclin (PGI2) incubated in Human Platelet Rich Plasma (PRP); in Platelet Poor Plasma (PPP) or in Krebs-Ringer-Bicarbonate (KRB) during different periods of time on contractions of bovine coronary arteries and on the ADP platelet aggregative capacity of human PRP, were explored. It was documented that incubates in PRP or in PPP retain an antiaggregatory activity at higher levels and during a longer time than in KRB. On the other hand, PGI2 incubates in KRB exhibited only a relaxing activity on isolated bovine coronary arteries, whereas when incubated in PRP or in PPP presented a biphasic influence. The initial effects (evoked by incubates of 30 minutes) were distinctly relaxing but those obtained with later incubates (60--150 minutes) stimulated clearly the resting basal tone of the arteries. The possibility that the human plasma might have an enzyme(s) able to transform prostacyclin into a more stable material with human antiaggregatory platelet function and bovine coronary contracting capacity is discussed.

Prostaglandin E release by rat medial basal hypothalamus in vitro. Inhibition by melatonin at submicromolar concentrations

Melatonin depressed PGE release by rat medial basal hypothalamus (MBH) in vitro at concentrations 10(-8) M or greater. The two-fold increase in PGE release induced by adding 10(-4) M norepinephrine to MBH explants was also impaired by melatonin at micromolar or greater concentrations. These data support the view that melatonin is a physiological inhibitor of PG synthesis in MBH.

Ovarian hormones inhibit the release of prostacyclin-like material from isolated rat uterus.

The influence of sex steroids on the production of prostacyclin (PGI2) like material by the isolated rat uterus incubated in a buffer medium was explored by monitoring its ability to inhibit ADP-induced platelet aggregation. Chopped uterine strips from rats in natural estrus can generate an unstable substance that inhibits platelet aggregation and suggest to be prostacyclin. This capacity was significantly enhanced in preparations from spayed animals. The injection of 17-beta estradiol; progesterone or both diminished the production of the prostacyclin-like material by the uterus from ovariectomized rats. The already existing notion that ovarian steroids are able to regulate the synthesis of stable prostaglandins is discussed together with the present results suggesting in addition a depressive effect of sex hormones on the uterine PGI2 synthetase system.

Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus.

The activity of matrix metalloproteinase (MMP)-9 was evaluated in placental tissue from healthy subjects (controls) and from patients with gestational and pre-existing diabetes mellitus (GDM and PDM, respectively). Compared with controls, MMP-9 activity was greater in placental tissue from patients with PDM and lower in placental tissue from patients with GDM. The modulatory role of nitric oxide (NO) and reactive oxygen species (ROS) on MMP-9 activity in placental tissue was evaluated. In healthy placenta, NO synthase inhibitors diminished MMP-9 activity, whereas NO donors enhanced it. The addition of xanthine/xanthine oxidase or hydrogen peroxide to placental incubates enhanced MMP-9 activity, while the addition of superoxide dismutase (SOD) diminished it. In placental tissue from patients with PDM, MMP-9 activity was stimulated by NO and by ROS. In placental tissue from patients with PDM, concentrations of nitrates/nitrites and thiobarbituric acid-reactive substances (TBARS) were enhanced, whereas SOD activity was decreased, suggesting that elevated concentrations of NO and ROS may be related to the enhanced MMP-9 concentrations found in these tissues. In placenta from GDM patients, in which a diminished concentration of MMP-9 were detected, nitrate/nitrite concentrations were increased, but placental MMP-9 activity did not change in the presence of either NO donors or inhibitors. The activity of MMP-9 in placental tissue from patients with GDM was stimulated by ROS donor systems and was inhibited by the addition of SOD; however, TBARS and SOD concentrations were unchanged in these tissues compared with controls. These findings demonstrate that placental MMP-9 activity is modulated by NO and ROS and that, in diabetic pathology, NO and ROS may determine changes in MMP-9 activity, which are probably involved in the structural and functional abnormalities of diabetic placental tissue.

Is the spontaneous motility of isolated rat uterus controlled by prostaglandin E

Variations in the spontaneous contractile activity during 6 hours following isolation of uterine horns from proestrus, metestrus and spayed rats, were explored. In estrus and metestrus preparations the contractions declined during 60 min and between 180--200 min a progressive spontaneous recovery (abolished by indomethacin) was observed up to 360 min. Uteri from proestrus and spayed animals exhibited a continuous depression without recovery during the whole experimental period. At 60 min, uterine horns from estrus animals (which showed a marked contractile decrement) released to the suspending medium significantly less prostaglandin E-like material than at 360 min, i.e. when contractions had almost completely recovered. No modification in the amount of prostaglandin F-like material was detected accompanying these spontaneous contractile variations. In the spayed group at 60 min of functioning (i.e. when the contractile impairment was significantly smaller than at a later time) the release of PGE was greater than at 360 min. These findings suggest a possible control of rat uterine contractions by PGE, rather than by PGF.

Role of nitric oxide on uterine and ovarian prostaglandin synthesis during luteolysis in the rat

In previous studies in our laboratory, we demonstrated that oxytocin (oxy) augmented prostaglandin F(2alpha) (PGF(2alpha)) synthesis via enhancing the uptake of Ca2+ by uterine tissue. On the other hand, we have shown that oxy enhances PGF(2alpha) synthesis in uterine and ovarian tissues during the corpus luteum (CL) regression in the rat. In the present study we explore the possible relation between endogenous nitric oxide (NO) and oxy on PGs synthesis during the luteolytic phase in the rat. The experiments were done in uterine and ovarian preparations isolated from pseudopregnant (psp) rats during the luteolytic phase. Tissues were incubated in vitro with 1)- oxy (50 mU/ml), 2)-NMMA (N(G)-monomethyl-L-arginine), a potent NOs inhibitor (300 uM), and 3)- both reagents (oxy + NMMA). NMMA decreases the synthesis of both PGs (PGE and PGF(2alpha)) and oxy enhances PGF(2alpha) synthesis in uterine and ovarian tissue. When reagents were used in combination (oxy + NMMA), we found different results in uterus and ovaries; i.e., in uterine tissue the NO inhibition did not affect the increase of PGF(2alpha) synthesis by oxy. Meanwhile, in ovaries the oxy effect over the PGF(2alpha) synthesis was not seen when NOs was inhibited. Probably oxy acts via different mechanisms on PGF(2alpha) synthesis in uterine and ovarian tissue. This assumption was confirmed when the NOs activity in both tissues (uterine and ovarian) was measured after oxy treatment. We found that oxy enhanced the NOs activity in ovarian tissues from psp rats but did not modify the enzyme activity in uterine tissue.

Metabolic factors regulating the generation of prostaglandins E1 and E2 by isolated rat uterus

The generation and output of PGE2 and PGE1 by isolated rat uterus incubated with or without glucose as well as the influence of different metabolic substrates and selective enzyme inhibitors on tissue formation and release of PGE material of the 1 and 2 series, were explored. The output of PGE2 was comparable with glucose, fructose, lactate or pyruvate as the substrate but diminished significantly in citrate-containing solution. In substrate-free media uteri released more PGE2 than in the presence of glucose or other substrates. The PGE1 liberated was similar in glucose, fructose, lactate or pyruvate and decreased significantly in citrate or without substrate. 2-deoxy-D-glucose (2-DG), fluoride or arsenite failed to alter significantly the release of PGE2 whereas 2,4-dinitrophenol (DNP) evoked augmentation. The uterus released less PGE1 after 2-DG or fluoride but not following the addition of DNP or arsenite. The results suggest that the synthesis and release of PGE2 and PGE1 in the isolated rat uterus appear to be selectively modulated by different tissue metabolic pathways.