Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000×g rat cerebellar supernatant preparation in a concentration-dependent manner (IC₅₀ value=12.9 μM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a K_i value of 2.57 μM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl₂/calmodulin, indicating that the presence of CaCl₂/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl₂/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.     

戊四唑癫痫大鼠脑组织一氧化氮含量和一氧化氮合酶活性变化

探讨一氧化氮在戊四唑癫病发机制中的作用。方法每天注射戊四唑建立在鼠癫痫模型,测定癫病发作后大鼠大脑皮质,海马一氧化氮和一氧化氮合酶活性变化,结果癫痫发作后海马ＮＯ含量和ＮＯＳ活性显著升高,结 戊四唑诱导的癫痫中具有致痫性。     

Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase

Background and purpose:

Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.

Experimental approach:

We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.

Key results:

Acute NOS inhibition by N_ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3^−/− mice but responses in NOS-3^−/− mice were enhanced by L-NAME.

Conclusions and implications:

Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3.     

Role of nitric oxide on insulin induced seizures in mice

The role of nitric oxide (NO) on acute hypoglycemia-induced seizures in mice was investigated using insulin as the hypoglycemic agent. The NO precursor L-arginine in the doses of 150, 500 and 750 mg/kg exhibited a dose-dependent protective effect against seizures induced by 8 mu/kg insulin. The NO synthase inhibitor (L-NMMA) at the doses of 50 and 100 mg/kg potentiated the subconvulsive doses of insulin (2 mu/kg). The onset, duration, number of seizures and the mortality were noted in a 2 hr study period. The results of this study suggest than NO plays an important protective role in acute hypoglycemia induced seizures which are known to occur through the activation of NMDA receptors.     

Synthesis, pharmacological activity and nitric oxide generation by nitrate derivatives of theophylline

Nitrates of theophylline derivatives - potential nitric oxide (NO) donors - were synthesized by esterification of 7-hydroxyalkyl theophylline derivatives with fuming nitric acid. The nitrates obtained were tested in-vitro in reactions with sulfydryl compounds at appropriately adjusted pH and temperature. Under the applied conditions, the synthesized compounds underwent decomposition to release NO, quantified using a polarographic method using a selective isolated (ISO-NO) sensor. The effects of dyphylline and proxyphylline and their new synthesized nitrates on arterial blood pressure (BP) were measured in spontaneously hypertensive (SH) rats. BP was measured in conscious SH rats using the tail-cuff method. Both short- and long-term administration of the xanthines tested significantly decreased systolic, diastolic and mean BP. The hypotensive effect of a single dose of nitrate dyphylline on mean BP was greater than that of the parent compound (P = 0.000012; P=0.000472 at 30 and 60 min post-dose, respectively), whereas proxyphylline and its nitrate derivative had similar activity. In rats treated with the tested compounds for 9 days twice daily, the decrease in BP persisted for at least 16 h after the last dose. Proxyphylline produced the most marked decrease in diastolic and mean BP. Among the xanthines examined, proxyphylline nitrate had the strongest hypotensive effect when administered in a single dose to animals pretreated with the same compound for 9 days. These results indicate that insertion of a nitrate group weakly modifies the hypotensive action of the studied xanthines in SH rats.     

Paroxetine-induced increase in metabolic end products of nitric oxide

Decreased production of endothelium-derived nitric oxide has been implicated in the pathogenesis of cardiovascular diseases. Metabolic end products of nitric oxide (NO(x)) are often used as markers for endothelial nitric oxide production in humans. Decreased endothelium-derived nitric oxide has been suggested to mediate some of the deleterious effects of conventional cardiovascular risk factors such as hypercholesterolemia, smoking, and physical inactivity. A substantial number of patients with cardiovascular diseases suffer from comorbid major depressive disorder, which is a predictor of a poorer cardiovascular outcome. Paroxetine is a first-line antidepressant and has been reported to decrease plasma NO(x), theoretically suggesting a potential deleterious effect on the cardiovascular system. We assessed the hypothesis that paroxetine would induce a decrease in plasma NO(x) in healthy volunteers. Plasma NO(x) levels were measured by chemiluminescence at baseline, after 8 weeks of paroxetine administration, and at postdiscontinuation. Contrary to our hypothesis, we found that paroxetine administration induced a significant increase in NO(x) that normalized after paroxetine discontinuation. It remains to be demonstrated that the paroxetine-induced increase in NO(x) is associated with a modification of the cardiovascular risk in patients with major depressive disorder.     

Paroxetine-induced increase in metabolic end products of nitric oxide

Decreased production of endothelium-derived nitric oxide (NO) has been implicated in the pathogenesis of cardiovascular diseases. Metabolic end products of nitric oxide (NOx) are often used as markers for endothelial NO production in humans. Decreased endothelium-derived NO has been suggested to mediate some of the deleterious effects of conventional cardiovascular risk factors such as hypercholesterolemia, smoking, and physical inactivity because they induce a decrease in plasma NOx. A substantial number of patients with cardiovascular diseases suffer from comorbid major depressive disorder, which is a predictor of a poorer cardiovascular outcome. Paroxetine is a first-line antidepressant and has been reported to decrease plasma NOx, theoretically suggesting a potential deleterious effect on the cardiovascular system. We assessed the hypothesis that paroxetine would induce a decrease in plasma NOx in healthy volunteers. Plasma NOx levels were measured by chemiluminescence at baseline, after 8 weeks of paroxetine administration, and at postdiscontinuation. Contrary to our hypothesis, we found that paroxetine administration induced a significant increase in plasma NOx that normalized after paroxetine discontinuation. It remains to be demonstrated that the paroxetine-induced increase in plasma NOx is associated with a modification of the cardiovascular risk in patients with major depressive disorder.     

Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors

The biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric-oxide synthase (NOS) and cyclooxygenase (COX) enzymes have been identified to date. Under normal circumstances, the constitutive isoforms of these enzymes (constitutive NOS and COX-1) are found in virtually all organs. Their presence accounts for the regulation of several important physiological effects (e.g. antiplatelet activity, vasodilation, and cytoprotection). On the other hand, in inflammatory setting, the inducible isoforms of these enzymes (inducible NOS and COX-2) are detected in a variety of cells, resulting in the production of large amounts of proinflammatory and cytotoxic NO and PGs. The release of NO and PGs by the inducible isoforms of NOS and COX has been associated with the pathological roles of these mediators in disease states as evidenced by the use of selective inhibitors. An important link between the NOS and COX pathways was made in 1993 by Salvemini and coworkers when they demonstrated that the enhanced release of PGs, which follows inflammatory mechanisms, was nearly entirely driven by NO. Such studies raised the possibility that COX enzymes represent important endogenous "receptor" targets for modulating the multifaceted roles of NO. Since then, numerous papers have been published extending the observation across various cellular systems and animal models of disease. Furthermore, other studies have highlighted the importance of such interaction in physiology as well as in the mechanism of action of drugs such as organic nitrates. More importantly, mechanistic studies of how NO switches on/off the PG/COX pathway have been undertaken and additional pathways through which NO modulates prostaglandin production unraveled. On the other hand, NO donors conjugated with COX inhibitors have recently found new interest in the understanding of NO/COX reciprocal interaction and potential clinical use. The purpose of this article is to cover the advances which have occurred over the years, and in particular, to summarize experimental data that outline how the discovery that NO modulates prostaglandin production has impacted and extended our understanding of these two systems in physiopathological events.     

Effect of nitric oxide gas on the generation of nitric oxide by isolated blood vessels: implications for inhalation therapy.

1. We have investigated, using rat aortic rings, whether exogenous nitric oxide (NO) gas affects the activity or expression of the inducible, Ca(2+)-independent NO synthase. 2. Incubation of rings with lipopolysaccharide (LPS, S. typhosa) for 6 h resulted in a gradual loss of tissue tone, a time-dependent reduction in constrictor response to phenylephrine and significant expression and activity of Ca(2+)-independent NO synthase. 3. Following incubation of LPS-treated rings with NO gas, the expression of inducible NO synthase mRNA was still observed, although the enzyme activity was significantly reduced and there was no reduction in the response to phenylephrine. 4. Therefore, NO gas can inhibit the action but not the induction of an NO synthase likely to play a role in inflammatory states such as adult respiratory distress syndrome (ARDS). 5. These observations may explain the rebound phenomenon observed in some ARDS patients following inhalation therapy with NO gas.     

Oxidation of N-hydroxyguanidine by nitric oxide and the possible generation of vasoactive species

It has been reported previously that the N-hydroxyguanidine function of N-hydroxy-L-arginine can react with nitric oxide (NO) to generate other species that can act as potent vasodilators with different biological lifetimes than NO. The identities of these species have yet to be determined. Therefore, we have studied the reaction between NO and N-hydroxyguanidine and determined that N-hydroxyguanidine is capable of reducing NO to yield nitrous oxide (N₂O) and possibly other nitroso species. It is likely that at least some of the N₂O formation in these reactions is due to the initial generation of nitroxyl (HNO). Since HNO has been shown to be a potent vasorelaxant, it is possible that some of the non-NO-mediated biological activity alluded to in previous studies was due to HNO and that other nitroso-species generated in the reaction may also contribute to the overall pharmacological activity by release of either NO or HNO.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

Bombesin-induced contractions of guinea pig lung strips are modulated by endogenous nitric oxide

We have investigated the effects of a nitric oxide (NO) biosynthesis inhibitor N^\sw-nitro-L-arginine methyl ester (l-NAME) on the bombesin-evoked contraction of guinea pig parenchymal lung strips. The bombesin-induced contractions of lung strips were significantly increased after l-NAME (300 M) pre-treatment. The maximal response was increased (P < 0.01) by 37% after l-NAME treatment when compared with the control group. The pD₂ value was not influenced by l-NAME pre-treatment. The enhancement of the bombesin-induced contraction caused by l-NAME was reversed by addition of an excess of the NO precursor-l-arginine (600 M) but not by the addition of its inactive enantiomer d-arginine (600 M). Like l-NAME, methylene blue (1 M), an agent that inhibits the soluble guanylyl cyclase activated by NO, significantly increased (P < 0.01) the maximal contraction induced by bombesin (183 ± 16 mg) when compared with the control group (141 ± 15 mg). When tested against other agonist-induced contractions, l-NAME did not change the responsiveness of parenchymal lung strips to bradykinin or carbachol but significantly increased the lung contraction induced by histamine. NO synthesis inhibition resulted in a pronounced increase in the bombesin-induced contraction of guinea-pig lung strips. Our results suggest that bombesin contributes to NO synthesis and release which then acts to reduce the contraction of the lungstrip in response to bombesin.     

In vitro generation of peroxynitrite by 2- and 4-hydroxyestrogens in the presence of nitric oxide

Estrogen metabolism is altered in most, if not all, breast cancer tumors. These alterations primarily lead to the formation of the catechol estrogen metabolites, 2- and 4-hydroxyestrogens, which can generate superoxide anion radicals (O(2)(*)(-)) through the redox cycling of semiquinone/quinone derivatives. In breast cancer cells, the activity of nitric oxide synthase is also frequently elevated, resulting in an increased level of exposure to nitric oxide ((*)NO). Since (*)NO rapidly reacts with O(2)(*)(-) to produce the peroxynitrite anion (ONOO(-)), this study was undertaken to determine whether ONOO(-) can be generated when 2- and 4-hydroxyestrogens are incubated in vitro with (*)NO donor compounds. Using dihydrorhodamine 123 as a specific probe for ONOO(-) formation, a ratio of 100 microM dipropylenetriamine NONOate (DPTA/NO) to 10 microM 4-hydroxyestradiol (4-OHE(2)) gave an optimal ONOO(-) production of 11.9 +/- 1.9 microM (mean +/- SD). Quantification of ONOO(-) was not modified by mannitol, supporting the idea that the hydroxyl radical was not involved. This production of ONOO(-) required the presence of the catechol structure of estrogen metabolites since all methoxyestrogens that were tested were inactive. Hydroxyestrogen metabolites derived from estradiol showed the same efficiency in producing ONOO(-) as those originating from estrone. With DPTA/NO, the 4-hydroxyestrogens generated 30-40% more ONOO(-) than the 2-hydroxyestrogens. Optimal production of ONOO(-) was assessed with DPTA/NO and diethylenetriamine NONOate (initial (*)NO generation rates of 0.76 and 0.08 microM min(-1), respectively). With faster (*)NO-releasing compounds, such as diethylamine NONOate and spermine NONOate, lower levels of ONOO(-) were detected. These data suggest that once the optimal concentration of (*)NO was obtained, the reaction between (*)NO and 4-OHE(2) was saturated. The excess of (*)NO would probably react with aqueous oxygen to form nitrite (NO(2)(-)). Since the third-order reaction rate for the reaction between 2(*)NO and O(2) is 2 x 10(6) M(-2) s(-1), it can therefore be suggested that the reaction between (*)NO and 4-OHE(2) occurs at a faster rate.     

Drastic increase in nitric oxide content in rat brain under halothane anesthesia revealed by EPR method

A drastic increase in nitric oxide (NO) content was revealed by the EPR method in rat brain cortex and cerebellum under halothane anesthesia. The NO scavenger diethyldithiocarbamate sodium salt (DETC) and ferrous citrate were injected into adult rats 30-60 min before anesthesia. Rats were anesthetized by inhalation of a halothane-oxygen mixture (1%, 1.5%, 2%, or 4%). After different times of anesthesia, rats were decapitated, and brain cortex and cerebellum were dissected, frozen in liquid nitrogen, and subjected to EPR spectroscopy. The concentration of NO was determined from the NO-Fe-DETC radical spectrum. In control animals, NO content in the cerebellum was only 68% of that in the cortex. We observed a time-dependent increase in NO content in the cortex and cerebellum of rats anesthetized with 1.5% halothane. In brain cortex, the NO level increased to six times that of waking animals after 30 min and remained at this level up to 60 min of anesthesia. In cerebellum the changes were less drastic, the NO level showing only a 2-fold increase. The same effect was produced by 1% and 2% halothane. Ketamine, chloral hydrate, and pentobarbital were used as reference drugs. None of these anesthetics produced effects similar to those of halothane. In ketamine-anesthetized rat brain, the NO content slightly decreased. Pentobarbital and chloral hydrate produced an insignificant increase in NO. Data are discussed in the context of possible interference of halothane in the regulation of nitric oxide synthase activity.     

Naphthazarin and methylnaphthazarin cause vascular dysfunction by impairment of endothelium-derived nitric oxide and increased superoxide anion generation.

The effects of the naphthoquinone analogue, naphthazarin (Nap), and its derivative, methylnaphthazarin (MetNap), on vascular reactivity were studied using isolated rat aortic rings and human umbilical vein endothelial cells (HUVECs). In this study, we determined vessel tension, nitric oxide (NO) formation, endothelial nitric oxide synthase (eNOS) activity, eNOS protein expression, and superoxide anion (O2*-) generation in an effort to evaluate the effect of Nap and MetNap on the impairment of the NO-mediated pathway. Lower concentrations of Nap (0.01-1 microM) and MetNap (1-10 microM) concentration-dependently enhanced phenylephrine (PE)-induced vasocontraction and abrogated acetylcholine (ACh)-induced vasorelaxation in an endothelium-dependent manner. On HUVECs, both Nap and MetNap concentration-dependently inhibited NO formation induced by A23187, and also partially inhibited nitric oxide synthase (NOS) activity. eNOS protein expression by HUVECs was not affected by treatment with Nap or MetNap, even within 24h. These data suggest that Nap and MetNap might act as inhibitors of nitric oxide synthesis in the endothelium. In addition, Nap and MetNap were also shown to generate O2*- on HUVECs with short-term treatment. We concluded that Nap and MetNap inhibited agonist-induced relaxation and induced vasocontraction in an endothelium-dependent manner, and these effects might have been due to modification of the NO content by inhibition of NOS activity and bioinactivation through O2*- generation.     

In situ generation of nitric oxide by myenteric neurons but not by mononuclear cells of the human colon

1. Production of nitric oxide (NO) is implicated in the pathogenesis of inflammatory bowel disease. However, the cells responsible for the production of NO in situ in the human colon remain unknown. 2. Surgical samples from 12 patients with ulcerative colitis, eight patients with Crohn's disease and 10 controls were studied. Possible generation of NO was visualized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity in human colon. Immunohistological staining for various NO synthase (NOS) isoforms (endothelial, neuronal and inducible), nitrotyrosine and interleukin-2 was also performed. 3. Reduced NADPH diaphorase activity was not found in lamina propria mononuclear cells, but was found in colonic epithelium, endothelium and myenteric neurons and their processes. 4. The NADPH-diaphorase activity positive processes were significantly less common in colon from patients with Crohn's disease compared with control colon. 5. Endothelial NOS was constitutively expressed on colonic endothelium. 6. Neuronal NOS was constitutively expressed on myenteric neurons. 7. Expression of inducible NOS (iNOS) was increased in the epithelium and endothelium of the colon of patients with ulcerative colitis. 8. No correlation was found between expression of iNOS and NADPH diaphorase activity. 9. Nitrotyrosine was expressed by lamina propria leucocytes, but not by epithelium. 10. Interleukin-2 was expressed on both leucocytes and myenteric neurons. 11. Colonic epithelium, endothelium and myenteric neurons synthesize NO. Myenteric neurons were principally responsible for NO production and NO may act as a neurotransmitter in the enteric nervous system.     

Semax prevents elevation of nitric oxide generation caused by incomplete global ischemia in the rat brain

A twofold increase in the nitric oxide (NO) production and a moderate increase in the content of secondary products of lipid peroxidation was observed in Wistar rats with incomplete global ischemia model induced by the bilateral occlusion of common carotid arteries. A clear correlation was observed between the NO content in the rat brain and the level of neurological disturbance manifestations in the ischemized animals. The synthetic peptide semax (a fragment of ACTH4-7 Pro-Gly-Pro) in a dose of 0.3 mg/kg prevented from the development of both neurological disturbances and excess NO production in the rat brain cortex.     

Chemical oxidation of N-hydroxyguanidine compounds. Release of nitric oxide, nitroxyl and possible relationship to the mechanism of biological nitric oxide generation.

N omega-Hydroxy-L-arginine was found to cause vasodilation in arginine-depleted rabbit aorta. It is, therefore, likely to be a biosynthetic intermediate in the conversion of arginine to nitric oxide in this tissue. N-Hydroxyalkylguanidine compounds, including N omega-hydroxy-L-arginine were oxidized with various oxidizing agents and examined for their ability to release nitric oxide. All oxidizing agents tested were capable of oxidizing the N-hydroxyguanidine function but only lead tetra-acetate (Pb(OAc)4) and potassium ferricyanide/hydrogen peroxide (K3FeCN6/H2O2) were capable of generating significant amounts of nitric oxide. Oxidation with K3FeCN6, lead oxide (PbO2) and silver carbonate (Ag2CO3) resulted instead in the release of nitrous oxide (N2O) presumably through the initial release of nitroxyl (HNO).