V. Novaro

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal and embryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A2 (PLA2) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF2 alpha production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF2 alpha decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF2 alpha. Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 microM) induced a decrease in PGE and PGF2 alpha production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.

Nitric oxide modulates placental prostanoid production from late pregnant non-insulin-dependent diabetic rat

Severe reproductive dysfunction has been described in non-insulin-dependent diabetes mellitus (NIDDM), correlated with high glucose levels in the plasma. We have characterized an abnormal prostanoid profile in tissues from NIDDM rats, and a tight correlation between nitric oxide (NO) levels and prostaglandin production. Likewise, we have determined that parturition is delayed in NIDDM rats compared to control animals. In order to characterize the events which precede delayed parturition in NIDDM rats, we evaluate (a) the arachidonic acid (AA) conversion in placental tissue obtained from control (day 21 and 22) and NIDDM (day 21, 22 and 23) late pregnant rats into prostaglandin E2 (PGE2) and F2alpha (PGF2alpha), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), and (b) NO synthase (NOS) activity in control and NIDDM late pregnant animals. Placental arachidonate conversion from control rats into different prostanoids, namely PGE2, PGF2alpha, and TXB2, is higher in day 22 than in day 21, and radioconversion from diabetic rats into PGE2, PGF22, TXB2 and 6-keto-PGF1alpha on day 23 is higher than in day 21 and 22. 6-keto-PGF1alpha is lower and TXB2 is higher in diabetic tissues than in control. Placental AA conversion of control diabetic tissues on the day of delivery is decreased by N(G) monomethyl-L-arginine (LNMMA) (600 mM), a well known NOS inhibitor, while prostanoid production remains unaltered on previous days. NOS activity is higher in control on day 22 when compared to day 21, and in diabetic on day 23 when compared to day 22 of pregnancy. We conclude that elevated NO placental levels are observed in control (day 22) and NIDDM (day 23) rats, and may increase placental prostaglandin production on the day of delivery.

Hyperglycemia promotes elevated generation of TXA2 in isolated rat uteri

The relationship between high glucose concentrations and arachidonic acid metabolism in uterine tissue from control and diabetic ovariectomized rats was evaluated. Uterine tissue from diabetic rats produced amounts of PGE2 and PGF2 alpha similar to controls, while a lower production of 6-keto-PGF1 alpha (indicating the production of prostacyclin) and a higher production of TXB2 (indicating the generation of TXA2) was found in the diabetic group. A group of diabetic rats was treated with phlorizin to diminish plasma glucose levels. Phlorizin treatment did not alter production of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha in the diabetic group. A diminished production of TXB2 was found in the treated diabetic uteri when compared to the non-treated diabetic group. Moreover, a positive correlation between plasma glucose levels and uterine TXB2 generation was observed. When control uterine tissue was exposed in vitro to high concentrations of glucose (22 mM) and compared to control tissue incubated in the presence of glucose 11 mM alterations in the generation of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were not found, but a higher production of TXB2 was observed and values were similar to those obtained in the diabetic tissue. Alteration in the production of the prostanoids evaluated were not observed when diabetic tissue was incubated in the presence of high concentrations of glucose. These results provide evidence of a direct relationship between plasma glucose levels and uterine production of TXA2.

Influence of Nitric Oxide Synthase and Kinin Antagonists on Metabolic Parameters in Chronic Streptozotocin-Induced Diabetes Mellitus

In vivo administration of HOE 140 (a new bradykinin receptor antagonist) and L-NAME (nitric oxide synthase inhibitor) was performed in chronic streptozotocin-diabetic rats. Basal increases (in umol.g dw-1) in liver (45.0 +/- 3.4.1) and uterine (40.0 +/- 2.95) triglyceride levels in diabetic animals vs control (liver: 34.0 +/- 3.87; uterus: 30.2 +/- 4.01) were partially prevented by L-NAME (p < 0.01), HOE 140 (p < 0.01) and L-NAME + HOE 140 (p < 0.01). High glycogen levels (in mg.g dw-1) observed in diabetic uterine tissue (3.07 +/- 0.90), and decreased glycogen content detected in diabetic liver (11.64 +/- 1.50) vs. control (uterus: 1.59 +/- 0.15, liver: 17.25 +/- 0.87) were unaffected. Uterine 14CO2 production from 14C-U-Glucose (in uCi.mg dw), which is lower in diabetic (35.0 +/- 5.12) than in control (50.12 +/- 4.54) tissues, was improved by HOE 140 (p < 0.05) and L-NAME+HOE 140 (p < 0.05), while hepatic glucose oxidation was not increased by the drugs. Glycemia levels were decreased in diabetic rats injected with L-NAME and L-NAME plus HOE 140. Pancreatic 6-Keto-prostaglandin F1 alpha to Thromboxane B2 ratio was lower in diabetic animals than in controls, and L-NAME and/or HOE 140 treatment prevented the decrement. These findings suggest that vasoactive compounds might prevent streptozotocin-induced damage in pancreatic tissue from chronic diabetic rats.

High glucose levels modulate eicosanoid production in uterine and placental tissue from non-insulin-dependent diabetic rats during late pregnancy

Severe uterine and placental disturbances have been described in diabetes pathology. The relative severity of these changes appears to correlate with high glucose levels in the plasma and incubating environment. In order to characterize changes in eicosanoid production we compared uterine and placental arachidonic acid conversion from control and non-insulin-dependent diabetes mellitus (NIDDM) rats on day 21 of pregnancy, into different prostanoids, namely PGE2, PGF22alpha, TXB2 (indicating the production of TXA2) and 6-keto-PGF1 (indicating the generation of PGI2). PGE2, PGF2alpha and TXB2 production was higher and 6-keto-PGF1alpha was similar in diabetic compared to control uteri. PLA2 activity was found diminished in the NIDDM uteri in comparison to control. A role for PLA2 diminution as a protective mechanism to avoid prostaglandin overproduction in uterine tissue from NIDDM rats is discussed. Placental tissues showed an increment in TXB2 generation and a decrease in 6-keto PGF1alpha level in diabetic rats when compared to control animals. Moreover, when control uterine tissue was incubated in the presence of elevated glucose concentrations (22 mM), similar generation of 6-keto PGF1alpha and elevated production of PGE2, PGF2alpha and TXB2 were found when compared to those incubated with glucose 11 mM. Placental TXB2 production was higher and 6-keto PGF1alpha was lower when control tissues were incubated in the presence of high glucose concentrations. However, high glucose was unable to modify uterine or placental prostanoid production in diabetic rats. We conclude that elevated glucose levels induced an abnormal prostanoid profile in control uteri and placenta, similar to those observed in non-insulin-dependent diabetic tissues.

Influence of prostaglandins on glucose transport in isolated rat uterus.

Glucose transport by uterine strips from ovariectomized estrogenized rats was explored. Sugar transport was significantly different from saccharose values (non-specific diffusion) only after 60 min of incubation. The addition of cytochalasin B demonstrated that we are measuring a specific mechanism for glucose transport. Insulin-enhanced sugar transport only at 0.5 or 0.25 U/ml prostaglandin E1 (PGE1), PGE2 and PGF2 alpha (10(-7) M) significantly improved glucose transport, but indomethacin (10(-6) M) failed in modifying this parameter in either control nor insulin-treated tissues. We did not observe an additive or synergistic action between PGE2 (10(-7) M) and insulin (used at maximal or submaximal concentration).

Influence of epoxyeicosatrienoic acids on uterine function

In spite of the large quantities of epoxyeicosatrienoic acids (EEts) released by reproductive tissues, their function has not yet been determined. In order to analyze the influence of epoxygenase products on isolated uterine function, Clotrimazole, a cytochrome P450 inhibitor was used. The drug decreased isolated rat uterine isometric developed tension (IDT) and frequency (FC). 14,15 EEt induced a contractile response when added at 10(11) M, 8,9 EEt and 11,12 EEt produced an increment of IDT when added to 10(-7) M and 5,6 EEt did not modify IDT values. A contractile stimulatory effect was observed when 14,15 EEt (10(-7) M) was added to a tissue bath preparation containing Clotrimazole (20 microM). On the other hand, uterine contractile response to 14,15 EEt addition was partially abolished by indomethacin (10(-6) M), a well known cyclooxygenase inhibitor. Uterine response to 5,6; 8,9 and 11,12 EEts was not modified by indomethacin. This is the first evidence of 14-15 EEt uterotonic properties, possibly exerted in part through the cyclooxygenase pathway.

Eicosanoid production by placental and amnion tissues from control and non-insulin-dependent diabetic rats. Influence of oxytocin in the incubating medium

Eicosanoid production by intrauterine tissues from control and neonatal-streptozotocin induced diabetic rats during late pregnancy was evaluated. In diabetic placenta the release of 6-keto-PGF1alpha was found diminished when compared to controls. In addition, LTB4 generation was increased in diabetic placenta. No alterations in the production of TXA2, PGE2, PGE1 and PGF2alpha was found when diabetic and control placenta were compared. In amnion tissue a decreased generation of 6-keto-PGF1alpha was observed in the diabetic group, but no alteration in any other eicosanoid evaluated was found. Oxytocin (5 mU/ml, in vitro), which increases prostaglandin synthesis in rabbit and human amnion tissues, did not modify eicosanoid generation in control rat amnion. In contrast, in diabetic amnion the presence of oxytocin further decreased the release of 6-keto-PGF1alpha and diminished PGE1 generation. The present results suggest that this mildly diabetic state induces alterations in eicosanoid production in intrauterine tissues, abnormalities probably enhanced during parturition, when endogenous concentrations of oxytocin are elevated.

Eicosanoid production and phospholipase A2 activity in uterine tissue from castrated rats with non-insulin dependent diabetes mellitus

In uterine tissue obtained from castrated control and non-insulin dependent diabetic (NIDDM) rats, eicosanoid production and its regulation by glucose levels and by the activity of phospholipase A2 (PLA2) was assessed. Basal outputs of prostaglandins (PGs) PGE2, PGE1, PGF2 alpha, 6-keto-PGF1 alpha (indicating the production of prostacyclin), thromboxane B2 (TXB2) (indicating the generation of TXA2) and leukotriene B4 (LTB4) were similar in control and NIDDM uterine preparations as assessed by RIA. When uterine conversion of labelled arachidonate into different prostanoids was evaluated, generation of 6-keto-PGF1 alpha, PGE2 and PGF2 alpha was similar in control and NIDDM uterine tissue, while TXB2 production was higher in the diabetic group. Moreover, when control tissue was incubated in the presence of elevated concentrations of glucose (22 mM) and compared to control tissue incubated in concentrations of glucose 11 mM, similar generation of 6-keto-PGF1 alpha, PGE2 and PGF2 alpha was observed, and higher concentrations of TXB2 were found, similar to those observed in diabetic uterine tissue. When NIDDM uterine tissue was incubated in the presence of glucose 22 mM, no difference in any prostanoid evaluated was observed when compared to values obtained in the presence of glucose 11 mM. In this work we have observed in NIDDM uterine tissue a normal TXA2 production when evaluated by RIA from endogenous arachidonic acid (AA) and a higher TXA2 generation from exogenous labelled AA. In addition PLA2 activity was found diminished in the NIDDM uteri in comparison to control uteri. A role of the diminished PLA2 as a protective mechanism that avoids TXA2 overproduction in uterine tissue from NIDDM rats is discussed.

The role of nitric oxide in the metabolism of labeled glucose in isolated rat uterus. Influence of 17β-estradiol

The influence of nitric oxide (NO) on the production of 14CO2 from labeled glucose in uteri isolated from ovariectomized-estrogenized rats was studied. Nitroprusside, an NO donor (NP), 200 μM increased the formation of labeled CO2 from [U-14C]glucose. This effect was blunted by hemoglobin (Hb) 20 μg/mL, an NO scavenger. The addition of N-monomethyl arginine (NMMA), an inhibitor of NO synthase decreased the stimulatory action of NP at 400 mM. Incubation of uterine strips in the presence of NP plus acetylsalicylic acid (ASA) 10−4 M (a cyclooxygenase inhibitor), inhibited the stimulatory action of NP on glucose metabolism. PGE2 (10−7 M) added to the incubation medium containing NP and ASA reversed the effect of the inhibitor. Neither NP nor Hb nor NMMA modified the 14CO2 production from labeled glucose in uterine strips from ovariectomized rats. The addition of NP to the incubating medium increased PGE accumulation by uterine strips from rats treated with estradiol, but not in ovariectomized animals. These results suggest that NO exerts a positive influence on glucose metabolism and PGE synthesis in isolated rat uteri from estrogenized animals.