Nitric oxide increases in the rat retina after continuous illumination.

Continuous illumination (CI) of the retina induces an oxidative stress followed by the degeneration of photoreceptors. This phenomenon may be partially related to the excessive production of nitric oxide (NO). In order to confirm this hypothesis, the aims of this work are to determine NO levels during the illumination of the retina by electron paramagnetic resonance (EPR), and if an increase of NO is found, to characterize the NOS isoform responsible of the increment by using Western blot. Sprague-Dawley rats were continuously illuminated with white light (12,000 lux) for 2, 24, 48 h, 5 and 7 days while control rats were maintained at light/dark cycles of 12/12 h. Using EPR, an increase of NO signal was observed in the light exposed retinas peaking at 24 h of CI. Western blot analysis showed the expression of iNOS in the illuminated retinas with a peak after 24 h of CI, but did not show significant differences of nNOS among illuminated and control retinas. In summary, there is an increase of NO during CI. Further studies will reveal whether this mechanism is responsible for light induced photoreceptor degeneration.

Peroxynitrites and impaired modulation of nitric oxide concentrations in embryos from diabetic rats during early organogenesis.

Maternal diabetes significantly increases the risk of congenital malformation, a syndrome known as diabetic embryopathy. Nitric oxide (NO), implicated in embryogenesis, has been found elevated in embryos from diabetic rats during organogenesis. The developmental signaling molecules endothelin-1 (ET-1) and 15-deoxy delta(12,14)prostaglandin J2 (15dPGJ2) downregulate embryonic NO levels. In the presence of NO and superoxide, formation of the potent oxidant peroxynitrite may occur. Therefore, we investigated peroxynitrite-induced damage, ET-1 and 15dPGJ2 concentrations, and the capability of ET-1, 15dPGJ2 and prostaglandin E2 (PGE2) to regulate NO production in embryos from severely diabetic rats (streptozotocin-induced before pregnancy). We found intense nitrotyrosine immunostaining (an index of peroxynitrite-induced damage) in neural folds, neural tube and developing heart of embryos from diabetic rats (P < 0.001 vs controls). We also found reduced ET-1 (P < 0.001) and 15dPGJ2 (P < 0.001) concentrations in embryos from diabetic rats when compared with controls. In addition, the inhibitory effect of ET-1, 15dPGJ2 and PGE2 on NO production found in control embryos was not observed in embryos from severely diabetic rats. In conclusion, both the demonstrated peroxynitrite-induced damage and the altered levels and function of multiple signaling molecules involved in the regulation of NO production provide supportive evidence of nitrosative stress in diabetic embryopathy.

15-Deoxy-Delta12,14-prostaglandin J2 and peroxisome proliferator-activated receptor gamma (PPARgamma) levels in term placental tissues from control and diabetic rats: modulatory effects of a PPARgamma agonist on nitridergic and lipid placental metabolism.

15-Deoxy-Delta(12,14)-prostaglandin J2 (15dPGJ2) is a peroxisome proliferator-activated receptor (3) (PPAR(3)) ligand that regulates lipid homeostasis and has anti-inflammatory properties in many cell types. We postulated that 15dPGJ2 may regulate lipid homeostasis and nitric oxide (NO) levels in term placental tissues and that alterations in these pathways may be involved in diabetes-induced placental derangements. In the present study, we observed that, in term placental tissues from streptozotocin-induced diabetic rats, 15dPGJ2 concentrations were decreased (83%) and immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was increased. In the presence of 15dPGJ2, concentrations of nitrates/nitrites (an index of NO production) were diminished (40%) in both control and diabetic rats, an effect that seems to be both dependent on and independent of PPAR(3) activation. Exogenous 15dPGJ2 did not modify lipid mass, but decreased the incorporation of (14)C-acetate into triacylglycerol (35%), cholesteryl ester (55%) and phospholipid (32%) in placenta from control rats, an effect that appears to be dependent on PPAR(3) activation. In contrast, the addition of 15dPGJ2 did not alter de novo lipid synthesis in diabetic rat placenta, which showed decreased levels of PPAR(3). We conclude that 15dPGJ2 modulates placental lipid metabolism and NO production. The concentration and function of 15dPGJ2 and concentrations of PPAR(3) were altered in placentas from diabetic rats, anomalies probably involved in diabetes-induced placental dysfunction.

Influence of peroxisome proliferator-activated receptor gamma activation by its endogenous ligand 15-deoxy Delta12,14 prostaglandin J2 on nitric oxide production in term placental tissues from diabetic women.

Diabetes induces alterations which condition placental remodelling. The levels of nitric oxide (NO) (a modulator of placental invasiveness, differentiation and proliferation) were higher in term placental explants from diabetic patients when compared to controls. Peroxisome proliferator-activated receptor gamma (PPARgamma) activation by its endogenous ligand 15-deoxy Delta(12,14)prostaglandin J(2) (15dPGJ(2)), is a differentiating factor of adipocytes and other cell types, such as trophoblasts. 15dPGJ(2) is also able to down-regulate NO production in different cell types. Our study evaluated the levels of 15dPGJ(2) and PPARgamma and the influence of PPARgamma activation by 15dPGJ(2) on the production of NO, in term placental tissues from control, pre-gestational and gestational diabetic patients. Our results showed that 15dPGJ(2) was present in human term placenta, and that its levels were diminished in gestational (P<0.05) and pre-gestational (P<0.002) diabetic women when compared to controls. Exogenous 15dPGJ(2) addition (2 x 10(-6) mol/l) down-regulated NO production in placenta from control (P<0.001) and pre-gestational diabetic (P<0.01) patients, but failed to do so in gestational diabetic women, whose placental PPARgamma expression was diminished in comparison to controls (P<0.001). As the exogenous activation of PPARgamma prevented NO overproduction in placenta from pre-gestational diabetic women, it may have the potential to improve fetal outcome in this pathology.

Elevated levels of endothelin-1 and prostaglandin E2 and their effect on nitric oxide generation in placental tissue from neonatal streptozotocin-induced diabetic rats.

Endothelin-1 (ET-1), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are regulators of feto-placental hemodynamics. In this study we explore the inter-regulatory pathways that modulate the levels of these vasoactive agents in control and neonatal streptozotocin-induced (n-stz) diabetic rat placenta. ET-1 levels are increased in diabetic placenta when compared to controls (P<0.001), and are strongly reduced by an NO synthase inhibitor (P<0.001). PGE(2) production is increased in diabetic placenta when compared to controls (P<0.01), but these levels are not modulated by ET-1. NO levels, similar in control and in diabetic placenta, are not influenced by PGE(2), but they are negatively modulated by ET-1 in both control (P<0.05) and diabetic (P<0.01) placenta. We conclude that rat placental ET-1 inhibits NO levels but does not modify PGE(2) concentrations. The elevated levels of ET-1 and PGE(2) in diabetic placenta, potent vasoconstrictors of placental vasculature, are probably related to the induction of placental insufficiency and fetal hypoxia in this pathology.

Modulation of nitric oxide concentration and lipid metabolism by 15-deoxy Delta12,14prostaglandin J2 in embryos from control and diabetic rats during early organogenesis.

The concentration of 15-deoxy Delta(12,14)PGJ(2) (15dPGJ(2)) and its effects on nitric oxide generation and neutral lipid in embryos from control and neonatal streptozotocin-induced (n-stz) diabetic rats during organogenesis were investigated. 15dPGJ(2) is produced in embryos during organogenesis, and its production is lower in embryos of n-stz diabetic rats than in embryos from control rats. Nitrate and nitrite concentrations were higher in embryos from n-stz diabetic rats and were reduced in the presence of 15dPGJ(2) both in embryos from control and diabetic rats. Thus, decreased 15dPGJ(2) concentrations in embryos from n-stz diabetic rats may be related to the high nitric oxide concentrations found in those embryos. Exogenous 15dPGJ(2) decreased cholesterol and cholesteryl ester concentrations in embryos from control and n-stz diabetic rats, and reduced triacylglycerol concentrations in control embryos. Incorporation of [(14)C]acetate into lipids showed decreased de novo synthesis of cholesteryl ester and triacylglycerides in embryos from n-stz diabetic rats compared with controls. Exogenous 15dPGJ(2) reduced the incorporation of [(14)C]acetate into triacylglycerides, cholesterol and cholesteryl ester in embryos from both control and n-stz diabetic rats. 15dPGJ(2) is present in embryos during organogenesis, and reduces embryonic nitric oxide production and lipid synthesis. The lower 15dPGJ(2) concentration in embryos from n-stz diabetic rats may result in developmental alterations in this diabetic model.

Involvement of inducible isoforms of COX and NOS in streptozotocin-pancreatic damage in the rat: interactions between nitridergic and prostanoid pathway.

Streptozotocin-induced pancreatic damage involves nitric oxide (NO) and prostaglandins (PGs) overproduction. In this work we aim to evaluate a putative relationship between the elevated NO levels and the altered prostanoid production in pancreatic tissue from streptozotocin-diabetic rats. Total NOS activity and nitrate/nitrite pancreatic levels in tissues from diabetic rats are decreased when the cyclooxygenase (COX) inhibitor indomethacin (INDO) is added to the incubating medium, while the addition of PGE(2)increases nitrate/nitrite production and NOS levels. INDO and PGE(2)selectively affect Ca(2+)-dependent NOS (iNOS) activity in diabetic tissues, and they have not been able to modify nitrate/nitrite levels, iNOS or Ca(2+)-dependent (cNOS) in control tissues. When the NOS inhibitor L-NMMA was present in the incubating medium, control pancreatic [(14)C]-Arachidonic Acid ([(14)C]-AA) conversion to 6-keto PGF(1 alpha)and to TXB(2)was lower, and PGF(2 alpha), PGE(2)and TXB(2)production from diabetic tissues diminished. The NO donors, spermine nonoate (SN) and SIN-1, enhanced TXB(2)levels in control tissues, while PGF(2 alpha), PGE(2)and TXB(2)levels from diabetic tissues were increased. PGE(2)production from control and diabetic tissues was assessed in the presence of the NO donor SN plus INDO or NS398, a specific PG synthase 2 inhibitor. When SN combined with INDO or NS398 was added, the increment of PGE(2)production was abolished by both inhibitors in equal amounts, indicating that the activating effect of nitric oxide is exerted on the inducible isoform of cyclooxygenase. In the diabetic rat, prostaglandins and NO seem to stimulate the generation of each other, suggesting a lack of regulatory mechanisms that control the levels of vasoactive substances in acute phase of beta-cell destruction.

Modulation of PGE2 generation in the diabetic embryo: effect of nitric oxide and superoxide dismutase.

In this work we assessed NO levels in the control and diabetic embryo during early organogenesis, and the ability of NO and SOD to modify embryonic PGE2 levels. Rats were made diabetic by steptozotocin (60 mg/kg) before mating. Diabetic embryos (day 10 of gestation) show increased nitrate/nitrite levels and enhanced NOS activity. The diabetic embryos release to the incubation medium increased amounts of PGE2 and have diminished PGE2 content. In the control embryo NO modulates PGE2 levels, but this modulatory pathway is not observed in the diabetic embryos. The diminished PGE2 content and the enhanced PGE2 release is prevented by SOD additions, both in the diabetic embryos and in control embryos cultured in the presence of diabetic serum (24 h culture, explantation day 9). The present results show that SOD additions prevent the abnormalities in the accumulation, production and release of PGE2 in diabetic embryos, probably related to the decrease in malformations.

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.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42 percent) and NO synthase (NOS) activity (50 percent) in diabetic but not in control samples. When NO production was blocked by N G-monomethyl-L-arginine (L-NMMA) (600 ÁM), SOD activity increased (15.21 +/- 1.23 vs 24.40 +/- 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 +/- 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 +/- 0.02 vs 0.20 +/- 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 +/- 0.05); spermine nonoate (100 ÁM) abolished the decrease in lipoperoxide level (0.70 +/- 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat.

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N(G)-monomethyl-L-arginine (L-NMMA) (600 microM), SOD activity increased (15.21 +/- 1.23 vs 24.40 +/- 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 +/- 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 +/- 0.02 vs 0.20 +/- 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 +/- 0.05); spermine nonoate (100 microM) abolished the decrease in lipoperoxide level (0.70 +/- 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

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.

Diminished PGE2 content, enhanced PGE2 release and defects in 3H-PGE2 transport in embryos from overtly diabetic rats.

Diminished PGE2 levels in diabetic embryos are related to the development of malformations, and thus the aim of the present study was to determine whether PGE2 levels are modified in rat embryos cultured in diabetic serum during organogenesis, and if PGE2 content and release, and 3H-PGE2 uptake and release, are altered in incubated diabetic embryos. Rats were made diabetic by steptozotocin (60 mg kg(-1)) before mating. Control rat embryos cultured for 24 h (explantation Day 9) in the presence of diabetic serum showed diminished PGE2 levels. When Day 10 diabetic embryos were incubated, embryo PGE2 levels were lower, but the PGE2 released to the incubation media was much higher than in controls. Uptake of 3H-PGE2 by diabetic embryos was initially enhanced (5-10 min), then reached similar levels to controls (20-100 min). Release of 3H-PGE2 previously incorporated during a 60-min incubation was greater in diabetic embryos than in controls. These results show diminished PGE2 content in both diabetic and normal embryos cultured in the presence of diabetic serum, but suggest that diabetic embryos have the capability to produce and release high levels of PGE2. The enhanced release of PGE2 is probably the result of transport abnormalities, and leads to the elevated PGE2 concentrations found in the incubating medium and to the diminished intraembryonic PGE2 levels that alter embryonic development.

Evolution of streptozotocin-pancreatic damage in the rat: modulatory effect of endothelins on the nitridergic and prostanoid pathway.

Many lines of evidence indicate that an increased pancreatic production of nitric oxide (NO) and prostaglandins (PGs) is found in the pancreas of streptozotocin-diabetic rats and that endothelins (ETs) are closely related to the nitridergic and prostanoid pathway in several tissues. In the present study the relationship between NO, ETs, and PGs has been explored in isolated pancreatic tissue from streptozotocin-diabetic rats. Pancreatic ET levels are higher in pancreatic tissues from diabetic (D) rats compared to control (C) animals. The addition of nitric oxide synthase (NOS) inhibitors (1 mM N(G)-nitro-l-arginine methyl ester, 600 microM N(G)-monomethyl-l-arginine) in the incubating medium reduces and NO donors (SIN-1, 300 microM spermine suppress, NONOate 100 microM) increases ET levels in pancreatic slices from C and D animals. PGE(2) (10(-7) M) increases and indomethacin (10(-6) M) decreases ET pancreatic production only in D but not in C tissues when added into the incubating bath. When tissues are incubated in the presence of endothelin 1 (ET-1) (10(-7) M), NOS activity is higher in C pancreas, while the ET-receptor antagonist bosentan (B) decreases NOS levels in D but not in C tissues. When pancreatic arachidonic acid (AA) conversion to prostaglandins was explored, ET-1 increased PGF(2alpha), PGE(2), and TXB(2) levels in C but not in D tissues. B abolishes TXB(2) increment due to the diabetic state, but failed in modulating AA conversion to 6-keto PGF(1alpha), PGF2(alpha) and PGE(2) in D pancreas. Our results show an alteration in AA metabolism, ET production, and NO increment associated with pancreatic damage due to streptozotocin.