Martha A. F. Gimeno

The nitric oxide synthase of mouse spermatozoa

Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 107 spermatozoa synthesized 7±2 pmol of l‐[14C]citrulline. Besides, l‐citrulline formation depended on the incubation time and on the concentration of l‐arginine present in the incubation medium. Different concentrations of N G‐nitro‐l‐arginine methyl ester (L‐NAME) but not aminoguanidine, inhibited l‐[14C]citrulline formation. Western‐blot analysis of solubilized sperm proteins revealed a unique band of M r=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

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.

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.

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.

Relationship between mouse uterine contractility, nitric oxide and prostaglandin production in early pregnancy

Despite the evidence for a functional role of nitric oxide (NO) in the regulation of uterine contractility in several species, there is little information about the effects of this gas on the mouse uterus. The aims of this study were to investigate if the NO relaxation pathway is present in mouse pregnant uterus and the relationship with the uterotonic prostaglandins (PGs E and F2alpha) production. We evaluated the effect of the treatment with a competitive nitric oxide synthase (NOs) inhibitor: N(G)-monomethyl-L-arginine on the spontaneous contractile activity and prostaglandin production on two different days of pregnancy: second day of pregnancy (preimplantation stage) and on the afternoon of the fifth day of pregnancy (postimplantation stage). We found that only on the fifth day of pregnancy did the inhibitor induce a highly significant isometric developed tension (IDT) and that this effect was maintained throughout the experiment. In order to evaluate if the generation of NO was also different between the two days of pregnancy, NOs activity was measured. Total NOs activity was significantly elevated during the postimplantation stage. We studied the interaction between the NO and cyclooxygenase (COX) pathways on the fifth day of pregnancy, and the data show no stimulation of PGs production by endogenous NO. In summary, we found that NO participates in the control of uterine contractility on the fifth day (a postimplantation stage) and that in this condition the NO was not able to elicit an increase in PGs production.

Nitric oxide mediates plateletactivating factor stimulatory action on uterine prostaglandin production

Accumulated evidence suggests that platelet-activating factor (PAF) may have a role in implantation by stimulating prostaglandin (PG) production. Since we had demonstrated that nitric oxide (NO) can increase uterine PGs, the aim of this study was to explore whether or not NO could mediate rat uterus responses to PAF on day 5 of gestation, when implantation takes place. Uterine motility was enhanced by PAF as compared to controls. This action was abolished by either the arginine analogue, N-monomethyl L-arginine (L-NMMA) or the cyclooxygenase inhibitor, indomethacin. On the other hand, NOS activity was detected in uterine strips and could be stimulated by PAF. The cyclooxygenase product PGE2 was also significantly stimulated by PAF. Inhibition of endogenous NO formation abolished the PAF effect on PG synthesis. Our results suggest that NO is an important intermediate in the interaction between PAF and PGs.

Effects of lysine clonixinate on cyclooxygenase I and II in rat lung and stomach preparations.

Lysine clonixinate (LC) is a drug of antiinflammatory antipyretic and analgesic activity that produces minor digestive side-effects. This fact induced us to think that LC is possibly a weak COX-1 inhibitor. In order to investigate our hypothesis we inhibited cyclooxygenase activity with LC or indomethacin (INDO) in rat lung and stomach obtained from rats treated with lipopolysacharide (LPS) and control rats. Rat lung preparations incubated with 14C-arachidonic acid synthesise mainly PGE2. LC at 2.5 and 4.1 x 10(-5) M does not modify the basal production of PGE2 (probably COX-1) but at 6.8 x 10(-5) M significantly inhibited PGE2 production (approximately 48.5% inhibition, P<0.001). On the other hand, INDO at 10(-6) inhibited the basal production of PGE2 by around 73%. In LPS-treated rats, the production of PGE2 was significantly higher than in the lungs of control rats, probably due to the induction of COX-2. The addition of LC at 2.7 and 4.1 x 10(-5) M recovered the control values of PGE2 inhibiting, probably only from COX-2 activity. LC at higher concentrations (6.8 x 10(-5) M) and INDO 10(-6) M inhibited PGE2 formed by COX-2 and also partly by COX-1 activity.

Ex vivo effects of lysine clonixinate on cyclooxygenases in rat lung and stomach preparations

Abstract—Lysine clonixinate (LC) is an anti-inflammatory, anti-pyretic and analgesic drug with minor digestive side effects, which might suggest a weak COX-1 inhibitor. The aim of this study focused on ex vivo effects of LC 40 mg/kg ip and indomethacin (INDO) 10 mg/kg ip in lung and stomach preparations of control rats and LPS-treated rats (5 mg/kg ip). The non-steroidal antiinflammatory drugs were administered concomitantly, following three hours and before one, two or three hours of LPS treatment. Tissues were weighed and incubated in 2 ml of Kress Ringer Bicarbonate buffer containing glucose (11 mM) under an atmosphere of 95% oxygen and 5% CO2. Approximately 200 mg of tissue were used for each determination; 0.25 μCi of 14C-arachidonic acid was added to each tube and the tissues were incubated for 60 min. Prostanoids were extracted from the incubation medium and separated by TLC. Results were expressed as a percentage of the total radioactivity of the plates (% of cpm on plate/100 mg ww). It was found that LC animals that were not given LPS did not modify the synthesis of PGE2; in lung and stomach tissues showing that did not inhibit COX-1 activity. However, LC inhibited clearly the synthesis of PGE2 in both preparations obtained from LPS-treated animals. The inhibition was shown when the rats were treated concomitantly, 3 h after or 1 or 2 h before the injection of LPS.