Strategies targeting the NO pathway to counteract extra-pulmonary manifestations of COPD: A systematic review and meta-analysis.

The clinical symptoms of chronic obstructive pulmonary disease (COPD) disease are accompanied by severely debilitating extra-pulmonary manifestations, including vascular dysfunction and hypertension. This systematic review evaluated the current evidence for several therapeutic interventions, targeting the nitric oxide (NO) pathway on hemodynamics and, secondarily, exercise capacity in patients with COPD. A comprehensive search on COPD and NO donors was performed on online databases. Of 934 initially found manuscripts, 27 were included in the review, and 16 in the meta-analysis. The analysis indicated inconsistent effects of dietary nitrate supplementation on exercise tolerance in COPD patients. Dietary nitrate supplementation decreased systolic (-3.7 ± 4.3 mmHg; p = 0.10) and diastolic blood pressure (BP; -2.6 ± 3.2 mmHg; p = 0.05) compared with placebo. When restricted to acute studies, a clinically relevant BP lowering effect of nitrate supplementation during diastole was observed (-4.7 ± 3.2 mmHg; n = 5; p = 0.05). In contrast, inhaled NO (iNO) at doses <20 ppm (+9.2 ± 11.3 mmHg) and 25-40 ppm (-5±2 mmHg) resulted in inconsistent effects on PaO2 (p = 0.48). Data on the effect of iNO on exercise capacity were too limited and inconsistent, but preliminary evidence suggests a possible benefit of iNO on pulmonary vascular resistance during exercise in severe COPD patients. Overall, the effects of acute dietary nitrate supplementation on BP may be of clinical relevance as an adjunct therapy and deserve further investigation in large sample size studies of COPD patients with and without cardiovascular comorbidities. iNO exerted inconsistent physiological effects, with the use of high doses posing safety risks.

Inorganic nitrate supplementation and blood flow restricted exercise tolerance in post-menopausal women.

Exercise tolerance appears to benefit most from dietary nitrate (NO3-) supplementation when muscle oxygen (O2) availability is low. Using a double-blind, randomized cross-over design, we tested the hypothesis that acute NO3- supplementation would improve blood flow restricted exercise duration in post-menopausal women, a population with reduced endogenous nitric oxide bioavailability. Thirteen women (57-76 yr) performed rhythmic isometric handgrip contractions (10% MVC, 30 per min) during progressive forearm blood flow restriction (upper arm cuff gradually inflated 20 mmHg each min) on three study visits, with 7-10 days between visits. Approximately one week following the first (familiarization) visit, participants consumed 140 ml of NO3- concentrated (9.7 mmol, 0.6 gm NO3-) or NO3-depleted beetroot juice (placebo) on separate days (≥7 days apart), with handgrip exercise beginning 100 min post-consumption. Handgrip force recordings were analyzed to determine if NO3- supplementation enhanced force development as blood flow restriction progressed. Nitrate supplementation increased plasma NO3- (16.2-fold) and NO2- (4.2-fold) and time to volitional fatigue (61.8 ± 56.5 s longer duration vs. placebo visit; p = 0.03). Nitrate supplementation increased the rate of force development as forearm muscle ischemia progressed (p = 0.023 between 50 and 75% of time to fatigue) with non-significant effects thereafter (p = 0.052). No effects of nitrate supplementation were observed for mean duration of contraction or relaxation rates (all p > 0.150). These results suggest that acute NO3- supplementation prolongs time-to-fatigue and speeds grip force development during progressive forearm muscle ischemia in postmenopausal women.

The potent radioprotective agents: Novel nitronyl nitroxide radical spin-labeled resveratrol derivatives.

It is commonly known that radiotherapy is still a key modality for treatment of cancer. Though this effect is desirable during radiotherapy, it leads to radiotoxicity on normal healthy cells. In the present research, we designed, synthesized and analyzed a series of nitronyl nitroxide radical (NITR) spin-labeled resveratrol (RES) derivatives. The cytotoxicity of the newly synthesized substances was tested on Jurkat T cells. The derivatives were studied as reactive oxygen species (ROS) scavenger to protect ionizing radiation of Jurkat T cells upon 6 Gy X-irradiation. The experimental results revealed that compound 2 and 3 could significantly alleviate the damage of Jurkat T cells, as evidenced by decreasing ROS production and restoring the cell apoptosis. Further mechanism investigations indicated that the radioprotective effects of the novel derivatives were largely associated with modulating the expression of apoptotic proteins including cIAP-1, cIAP-2, cytochrome c, caspase-3 and caspase-9. Based on the experimental result, we disclosed that the novel NITR spin-labeled RES derivatives exhibit the potential to be used as the novel radioprotective candidates to ameliorate the injury induced by ionizing radiation.

Improvement in storage quality of postharvest tomato fruits by nitroxyl liposomes treatment.

Nitroxyl (HNO) has attracted much attention due to its unique biological activity. To investigate the preservation effect of HNO on fruits, a nitroxyl liposome based on 1-nitrosocyclohexyl acetate was prepared and characterized by infrared spectroscopy and transmission electron microscopy. The optimal preparation conditions were explored, and then HNO liposomes were prepared under the optimal conditions to study the effect of HNO liposomes on postharvest quality of tomatoes. The tomato fruits were treated with different concentrations (0, 5, 10, 15 and 20 µmol L-1) of HNO liposomes and stored at room temperature. The results indicated that treatment with HNO liposomes can more effectively delay the browning and slow down the decrease in lightness of tomatoes. Additionally, HNO liposomes can reduce the activity of PPO and POD, inhibit the increase of MDA and total phenol content. These results suggest that treatment with HNO liposomes can effectively preserve the quality of tomatoes.

Synthesis, antioxidant properties and neuroprotection of α-phenyl-tert-butylnitrone derived HomoBisNitrones in in vitro and in vivo ischemia models.

We herein report the synthesis, antioxidant power and neuroprotective properties of nine homo-bis-nitrones HBNs 1-9 as alpha-phenyl-N-tert-butylnitrone (PBN) analogues for stroke therapy. In vitro neuroprotection studies of HBNs 1-9 against Oligomycin A/Rotenone and in an oxygen-glucose-deprivation model of ischemia in human neuroblastoma cell cultures, indicate that (1Z,1'Z)-1,1'-(1,3-phenylene)bis(N-benzylmethanimine oxide) (HBN6) is a potent neuroprotective agent that prevents the decrease in neuronal metabolic activity (EC50 = 1.24 ± 0.39 µM) as well as necrotic and apoptotic cell death. HBN6 shows strong hydroxyl radical scavenger power (81%), and capacity to decrease superoxide production in human neuroblastoma cell cultures (maximal activity = 95.8 ± 3.6%), values significantly superior to the neuroprotective and antioxidant properties of the parent PBN. The higher neuroprotective ability of HBN6 has been rationalized by means of Density Functional Theory calculations. Calculated physicochemical and ADME properties confirmed HBN6 as a hit-agent showing suitable drug-like properties. Finally, the contribution of HBN6 to brain damage prevention was confirmed in a permanent MCAO setting by assessing infarct volume outcome 48 h after stroke in drug administered experimental animals, which provides evidence of a significant reduction of the brain lesion size and strongly suggests that HBN6 is a potential neuroprotective agent against stroke.

Wavelength-dependence of vasodilation and NO release from S-nitrosothiols and dinitrosyl iron complexes by far red/near infrared light.

Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830 nm) exhibiting reduced potency when compared to 670 nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670 nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740 nm and 830 nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670 nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.

Influence of dietary nitrate supplementation on lung function and exercise gas exchange in COPD patients.

BACKGROUND: During exercise as pulmonary blood flow rises, pulmonary capillary blood volume increases and gas exchange surface area expands through distention and recruitment. We have previously demonstrated that pulmonary capillary recruitment is limited in COPD patients with poorer exercise tolerance. Hypoxia and endothelial dysfunction lead to pulmonary vascular dysregulation possibly in part related to nitric oxide related pathways. PURPOSE: To determine if increasing dietary nitrate might influence lung surface area for gas exchange and subsequently impact exercise performance. METHODS: Subjects had stable, medically treated COPD (n = 25), gave informed consent, filled out the St George Respiratory Questionnaire (SGRQ), had a baseline blood draw for Hgb, performed spirometry, and had exhaled nitric oxide (exNO) measured. Then they performed the intra-breath (IB) technique for lung diffusing capacity for carbon monoxide (DLCO) as well as pulmonary blood flow (Qc). Subsequently they completed a progressive semi-recumbent cycle ergometry test to exhaustion with measures of oxygen saturation (SpO2) and expired gases along with DLCO and Qc measured during the 1st work load only. Subjects were randomized to nitrate supplement group (beetroot juice) or placebo group (black currant juice) for 8 days and returned for repeat of the above protocol. RESULTS: Exhaled nitric oxide levels rose >200% in the nitrate group (p < 0.05) with minimal change in placebo group. The SGRQ suggested a small fall in perceived symptom limitation in the nitrate group, but no measure of resting pulmonary function differed post nitrate supplementation. With exercise, there was no influence of nitrate supplementation on peak VO2 or other measures of respiratory gas exchange. There was a tendency for the exercise DLCO to increase slightly in the nitrate group with a trend towards a rise in the DLCO/Qc relationship (p = 0.08) but not in the placebo group. The only other significant finding was a fall in the exercise blood pressure in the nitrate group, but not placebo group (p < 0.05). CONCLUSION: Despite evidence of a rise in exhaled nitric oxide levels with nitrate supplementation, there was minimal evidence for improvement in exercise performance or pulmonary gas exchange surface area in a stable medically treated COPD population.

LDL dinitrosyl iron complex: A new transferrin-independent route for iron delivery in hepatocytes.

In view of the interrelations between NO, Fe, and LDL in the cardiovascular system it appears interesting to find out, if the lipoprotein particles undergo the process of iron-nitrosylation, commonly observed for other proteins and what is the biological fate of iron-nitrosylated LDL particles. Iron-nitrosylated LDL preparation containing Fe(NO)2 motif (DNICLDL) was obtained and characterized for the first time. In order to test its interactions with potential target cells, DNICLDL was administered to the hepatoma HepG2 cells. The effects were referred to those induced by native LDL (nLDL) and oxidized LDL (oxLDL) particles. DNICLDL administration considerably increased total iron content in the studied cell line, but did not influence the level of calcein-chelatable ions. DNICLDL was found to be low toxic to cells. The study suggests that DNICLDL might be a potential transducer of iron. © 2017 BioFactors, 44(2):192-201, 2018.

Neuroprotective Effect and Mechanism of Action of Tetramethylpyrazine Nitrone for Ischemic Stroke Therapy.

Our previous studies demonstrated that the multifunctional agent TBN, a derivative of tetramethylpyrazine armed with a nitrone moiety, displayed high therapeutic efficacy in experimental ischemic stroke models. However, its molecular mechanisms of action underlying the neuroprotective effect need further exploration. In the present study, we found that TBN had significant activities scavenging free radicals such as ·OH, O 2·- and ONOO-, inhibiting Ca2+ overload, maintaining mitochondrial function and preventing neuronal damage in primary cortical cultures. Further, TBN was effective in reducing brain infarction and ameliorating impairment of behavioral functions in the permanent middle cerebral artery occlusion (p-MCAo) rat model. TBN down-regulated the expression of pro-apoptotic factors Bax, while up-regulated the expression of anti-apoptotic factor Bcl-2 and increased the expression of pro-survival factors including p-Akt and p-GSK3ß in the peri-infarct cortex of p-MCAo rats. In addition, LY-294002 (a PI3K inhibitor) and MK2206 (an Akt inhibitor) significantly blocked the protective effect of TBN against OGD-induced death of cortical neurons. Taken together, the multifunctional mechanisms including scavenging free radicals, blocking calcium overload, maintaining mitochondrial function and activating the PI3K/Akt/p-GSK3ß cell survival pathway were possibly involved in the neuroprotective effects of TBN, making it a promising clinical candidate for the treatment of ischemic stroke.

Evaluation of mitochondrial bioenergetics, dynamics, endoplasmic reticulum-mitochondria crosstalk, and reactive oxygen species in fibroblasts from patients with complex I deficiency.

Mitochondrial complex I (CI) deficiency is the most frequent cause of oxidative phosphorylation (OXPHOS) disorders in humans. In order to benchmark the effects of CI deficiency on mitochondrial bioenergetics and dynamics, respiratory chain (RC) and endoplasmic reticulum (ER)-mitochondria communication, and superoxide production, fibroblasts from patients with mutations in the ND6, NDUFV1 or ACAD9 genes were analyzed. Fatty acid metabolism, basal and maximal respiration, mitochondrial membrane potential, and ATP levels were decreased. Changes in proteins involved in mitochondrial dynamics were detected in various combinations in each cell line, while variable changes in RC components were observed. ACAD9 deficient cells exhibited an increase in RC complex subunits and DDIT3, an ER stress marker. The level of proteins involved in ER-mitochondria communication was decreased in ND6 and ACAD9 deficient cells. |ΔΨ| and cell viability were further decreased in all cell lines. These findings suggest that disruption of mitochondrial bioenergetics and dynamics, ER-mitochondria crosstalk, and increased superoxide contribute to the pathophysiology in patients with ACAD9 deficiency. Furthermore, treatment of ACAD9 deficient cells with JP4-039, a novel mitochondria-targeted reactive oxygen species, electron and radical scavenger, decreased superoxide level and increased basal and maximal respiratory rate, identifying a potential therapeutic intervention opportunity in CI deficiency.

Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis.

Nitric oxide (NO) is an important cellular signaling molecule that modulates various physiological activities. Angiogenesis-promoting activities of NO-donor drugs have been explored in both experimental and clinical studies. In this study, a structurally well characterized and water-soluble neutral {Fe(NO)2}(9) DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) was synthesized to serve as a NO-donor species. The antitumor activity of DNIC 2 was determined by MTT assay, confocal imaging, and Annexin-V/PI staining. The IC50 values of DNIC 2 were 18.8, 42.9, and 38.6 µM for PC-3, SKBR-3, and CRL5866 tumor cells, respectively. Moreover, DNIC 2 promoted apoptotic cell death via activation of apoptosis-associated proteins and inhibition of survival associated proteins. In particular, DNIC 2 treatment suppressed PC-3 tumor growth by 2.34- and 19.3-fold at 7 and 21 days, in comparison with the control group. These results indicate that water-soluble DNIC 2 may serve as a promising drug for cancer therapy.

Effect of CO, NOx and SO2 on ROS production, photosynthesis and ascorbate-glutathione pathway to induce Fragaria×annasa as a hyperaccumulator.

A study was conducted to determine the effect of carbon monoxide (CO), nitroxide (NOx) and sulfur dioxide (SO2) on ROS production, photosynthesis and ascorbate-glutathione pathway in strawberry plants. The results showed that both singlet oxygen (O2(-1)) and hydrogen peroxide (H2O2) content increased in CO, NOx and SO2 treated strawberry leaves. A drastic reduction of primary metabolism of plants (photosynthesis), with the closure of stomata, resulted in a reduction of protein, carbohydrate and sucrose content due to production of reactive oxygen species (ROS) under prolonged exposure of gas stress. The resulting antioxidant enzymes were increased under a low dose of gas stress, whereas they were decreased due to a high dose of gas stress. Our results indicate that increased ROS may act as a signal to induce defense responses to CO, NOx and SO2 gas stress. The increased level of antioxidant enzymes plays a significant role in plant protection due to which strawberry plants can be used as a hyperaccumulator to maintain environmental pollution, however, the defense capacity cannot sufficiently alleviate oxidative damage under prolonged exposure of CO, NOx and SO2 stress.

Therapeutic effects of multifunctional tetramethylpyrazine nitrone on models of Parkinson's disease in vitro and in vivo.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Although the etiology of PD is not completely understood, it is well-documented that oxidative stress and Ca(2+)-mediated cellular damage play important roles in the progression of PD. 2-[[(1,1-Dimethylethyl)oxidoimino]-methyl]-3,5,6-trimethylpyrazine (TBN), a novel nitrone derivative of tetramethylpyrazine, has shown significant therapeutic effects in stroke models due to its multiple functions, including calcium overload blockade and free radical-scavenging. In this study, we investigated the neuroprotective and neurorescue effects of TBN on various in vitro and in vivo models of PD and explored its possible mechanisms of action. The results show that TBN exerted significant neuroprotection on 1-methyl-4-phenylpyridinium (MPP(+))-induced damage in SH-SY5Y cells and primary dopaminergic neurons, as well as on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuron loss in zebrafish (TBN and MPTP were added simultaneously into the fish embryo medium and the treatment period was 48 h). In the MPTP-induced mouse and 6-hydroxydopamine (6-OHDA)-induced rat PD models, TBN administrated orally twice daily for 14 d (3 d post-MPTP lesion in mice and 7 d post-6-OHDA lesion in rats) exhibited remarkable neurorescue effects to increase the number of dopaminergic neurons. In addition, TBN improved apomorphine-induced rotational behavior in the 6-OHDA-lesioned PD rats. TBN suppressed the MPP(+)-induced intracellular reactive oxygen species (ROS) in SH-SY5Y cells, increased the superoxide dismutase (SOD) activity and glutathione (GSH) concentration in the substantial nigra of MPTP-treated mice. These data indicate that TBN protects and rescues dopaminergic neurons from MPP(+) and MPTP/6-OHDA-induced damage by reducing ROS and increasing cellular antioxidative defense capability.

Effects of curcumin on nitrosyl-iron complex-mediated DNA cleavage and cytotoxicity.

Combination therapy aims to improve the pharmaceutical efficacy of different drugs, thus lowering the dosages used and reducing the side effects. However, interactions between individual drugs may also occur and lead to uncertain consequences. This study demonstrated that curcumin, a natural phenolic compound found in the rhizomes of turmeric, could either inhibit or enhance DNA cleavage caused by the synthetic nitrosyl-iron complex NC10 ([Fe2(C2H5OS)2(NO)4]). Without UV irradiation, higher concentrations of curcumin protected DNA from being cleaved by NC10. Conversely, in the presence of lower concentrations of curcumin (< 5 µM), cleaved DNA increased by raising curcumin concentrations. After UV irradiation, the DNA protective effect of curcumin decreased while the enhancing DNA cleavage effect of curcumin remained. UV/visible spectroscopy analysis showed that curcumin is associated with the iron of NC10, suggesting the formation of curcumin-Fe complexes. Furthermore, a cytotoxicity assay revealed that cotreatment of NC10 and curcumin had synergetic effects on the growth inhibition of mouse melanoma B16-F10 cells. To our knowledge, this is the first study of the cotreatment of curcumin with inorganic compounds that showed synergistic cytotoxicity.

[In vitro cytotoxicity of nitroxyl radicals with respect to tumor and diploid human cells and estimation of their antiviral activity].

Thirty nine water soluble nitroxyl radicals of various classes, belonging to piperidine, pyrrolidine and imidazolidine series were synthesized. Twenty seven of them were cytotoxic in vitro with respect to the tumor cell culture A431. The CC50 of the most active nitroxyl radicals with respect to cells SW480 and A431 was within 0.16-2.5 mM at the selectivity index of 3.91-7.81 in relation to cytotoxicity of the compounds for the cells of the normal L68 phenotype and tumor cells. The tests on the antiviral activity showed that 16 out of 22 nitroxyl radicals had antiviral activity in Vero cell culture with respect to the West Nile virus and Herpes simplex virus of type II respectively. The EC50 ranged within 0.09-3.45 mM. Some of the nitroxyl radicals had only antiviral activity, but a number of the compounds had both cytotoxic properties and antiviral activity.

[The study of hypotensive action of dinitrosyl-iron complex with glutathione containing drug oxacom in healthy volunteers].

On the basis of earlier executed studies of hypotensive effect of dinitrosyl iron complexes (DNIC) with glutathione, the drug has been created in industrial conditions named oxacom. Preliminary pharmacological studies of oxacom have not revealed negative qualities. The drug has been now tested in 14 healthy men in whom at single intravenous introduction it caused typical response - a decrease of diastolic as well as systolic arterial pressure on 24-27 mmHg through 3-4 min with subsequent very slow restoration in 8-10 hours. The heart rate after initial rise was quickly normalized. Echocardiography revealed unaltered cardiac output in spite of reduced cardiac filling by 28%. The multilateral analysis of clinical and biochemical data has revealed an absence of essential alterations which could lead to pathological consequences. The drug is recommended for carrying out of the second phase of clinical trial. The comparative study of the efficiency of hypotensive action of oxacom, S-nitrosoglutathione (GS-NO) and sodium nitrite (NO2) in rats has shown that the duration of effect was the greatest at oxacom action.

Synthesis, characterization and biological evaluation of mononuclear Co(II), Ni(II), Cu(II) and Pd(II) complexes with new N2O2 Schiff base ligands.

New tetradentate N(2)O(2) donor Schiff bases and their mononuclear Co(II), Ni(II), Cu(II), and Pd(II) complexes were synthesized and characterized extensively by IR, (1)H-, (13)C-NMR, mass, ESR, conductivity measurements, elemental and thermal analysis. Specifically the magnetic and electronic spectral measurements demonstrate the octahedral structures of cobalt(II), nickel(II) complexes and square planar geometries of copper(II), palladium(II) complexes. All the ligands and complexes were screened for their in vitro antibacterial activity against two gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) and two gram-negative bacteria (Escherichia coli, Klebsiella pneumonia). In this study, Pd(II) complexes exhibited potent antibacterial activity against B. subtilis, S. aureus whereas other metal complexes also exerted good activity towards all tested strains even than standard drugs streptomycin and ampicillin.

Anti-cancer activity of nitrones in the Apc(Min/+) model of colorectal cancer.

Abstract The nitrones of alpha-phenyl-tert-butyl nitrone (PBN) and 4-hydroxyl-PBN (4-OH-PBN) that have anti-cancer activity in models of liver cancer and glioblastomas were tested in the ApcMin/+ mouse model. Mice were administered PBN and 4-OH-PBN in drinking water and intestinal tumour size and number assessed after 3-4 months. Throughout the experiment, contrast-enhanced magnetic resonance imaging (MRI) was used to monitor colon tumours. MRI data showed a time-dependent significant increase in total colonic signal intensity in sham-treated mice, but a significant decrease for PBN-treated mice and slight decrease for 4-OHPBN treated mice, probably due to the limited water solubility of 4-OH-PBN. Final pathological and percentage survival data agreed with the MRI data. PBN had little effect on oxaliplatin-mediated killing of HCT116 colon cancer cells and caused only a slight decrease in the amount of active fraction caspase 3 in oxaliplatin-treated cells. PBN has significant anti-cancer activity in this model of intestinal neoplasia.

Nitric oxide physiological responses and delivery mechanisms probed by water-soluble Roussin's red ester and {Fe(NO)2}10 DNIC.

Dinitrosyl-iron complexes (DNICs) are stable carriers for nitric oxide (NO), an important biological signaling molecule and regulator. However, the insolubility of synthetic DNICs, such as Roussin's red ester (RRE), in water has impaired efforts to unravel their biological functions. Here, we report a water-soluble and structurally well-characterized RRE [Fe(mu-SC2H4COOH)(NO)2]2 (DNIC-1) and a {Fe(NO)2}(10) DNIC [(PPh2(Ph-3-SO3Na))2Fe(NO)2] (DNIC-2), their NO-induced protein regulation, and their cellular uptake mechanism using immortalized vascular endothelial cells as a model. Compared with the most common NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), the in vitro NO release assay showed that both DNICs acted as much slower yet higher stoichiometric NO-release agents with low cytotoxicity (IC50 > 1 mM). Furthermore, L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose- and time-dependent manner. EPR spectroscopic analysis showed, for the first time, that intact DNIC-1 can either diffuse or be transported into cells independently and can transform to either paramagnetic protein bound DNIC in the presence of serum or [DNIC-(Cys)2] with excess L-cysteine under serum-free conditions. Both DNICs subsequently induced NO-dependent upregulation of cellular heat shock protein 70 and in vivo protein S-nitrosylation. We conclude that both novel water-soluble DNICs have potential to release physiologically relevant quantities of NO and can be a good model for deciphering how iron-sulfur-nitrosyl compounds permeate into the cell membrane and for elucidating their physiological significance.

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations.

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.