Antidiabetes drug metformin is a donor of nitric oxide: EPR measurement of efficiency

It is shown that metformin, which is a drug used for treatment of type 2 diabetes mellitus, is metabolized in vivo in the intestine and liver of mice with the release of nitric oxide. Subsequently the released nitric oxide forms paramagnetic mono- and dinitrosyl iron complexes which can be registered by EPR. It is suggested that nitric oxide is responsible for the multifarious therapeutic action of metformin such as lowering of blood glucose level, reduction of arterial hypertension, and other biological effects.     

Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor

The aqueous decay and concomitant release of nitric oxide (*NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO2) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of *NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO2 decay and *NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of *NO from LNO2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodide-based analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of *NO via pathways subject to regulation by the relative distribution of LNO2 to hydrophobic versus aqueous microenvironments.     

ESR characterization of a novel spin-trapping agent, 15N-labeled N-tert-butyl-alpha-phenylnitrone,as a nitric oxide donor

We previously found that one of the pharmacological effects of N-tert-butyl-alpha-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50% stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.     

The pharmacology of a new nitric oxide donor: B-NOD

We describe here a new compound, B-NOD, which, in vitro and in situ, releases nitric oxide (NO). Its activity in situ persists for more than 7 h, it does not cause a fall in blood pressure or an increase in heart rate and can be orally administered. It increases cyclic guanosine monophosphate (cGMP) and prevents platelet aggregation. In vitro, its release of NO is augmented by the presence of living cells (blood platelets). B-NOD may be useful in a number of clinical conditions in which prolonged release of NO without hemodynamic effects are desirable. A combination of aspirin with B-NOD could be formulated in which the individual concentrations of aspirin and B-NOD may be useful in the long-term treatment of coronary artery disease and in clinical situations in which long-term release of NO may be beneficial.     

Reaction between imidazolineoxil N-oxide (carboxy-ptio) and nitric oxide released from cultured endothelial cells: Quantitative measurement of nitric oxide by ESR spectrometry

The performance of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide for the monitoring agent of nitric oxide was investigated. The agent (125–500 μM) was mixed with equal volume of nitric oxide solution, and aliquots of the mixture were applied to ESR spectroscopy. ESR spectra of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl, a product of the agent reacted with nitric oxide, were observed. A linear relationship was observed between the amplitude of the signal and concentrations of nitric oxide up to 80 μM. Endothelial cells cultured on microcarries were packed in a column, perfused with Krebs solutions and the effluent was mixed to the agent. The same ESR spectra were obtained and amplitude of the signal was increased by bradykinin (3–300nM), decreased by preincubation of N^G-monomethyl-L-arginine (3–100 μM) and reversed by following incubation of L-arginine (100 μM).     

Furoxan nitric oxide donor coupled chrysin derivatives: synthesis and vasculoprotection

A series of furoxan-based nitric oxide-releasing chrysin derivatives were synthesized. Pharmacological assays indicated that all chrysin derivatives exhibited in vitro inhibitory activities against aldose reductase and advanced glycation end-product formation. Some chrysin derivatives were also found to increase the glucose consumption of HepG2 cells. Furthermore, the compounds released a low amount of NO in the presence of l-cysteine (range from 0.20% to 1.89%). These hybrid furoxan-based NO donor chrysin derivatives offer a mutual prodrug design concept for the development of therapeutic or preventive agents for vascular complications due to diabetes.     

Analysis of nitric oxide donor effectiveness in resistance vessels

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius; 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery; and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Celecoxib prodrugs possessing a diazen-1-ium-1,2-diolate nitric oxide donor moiety: Synthesis,biological evaluation and nitric oxide release studies

A new class of anti-inflammatory (AI) cupferron prodrugs was synthesized wherein a diazen-1-ium-1,2-diolato ammonium salt, and its O²-methyl and O²-acetoxyethyl derivatives, nitric oxide (NO) donor moieties were attached directly to an aryl carbon on a celecoxib template. The percentage of NO released from the O²-methyl and O²-acetoxyethyl compounds was higher (18.0–37.8% of the theoretical maximal release of one molecule of NO/molecule of the parent compound) upon incubation in the presence of rat serum, relative to incubation with phosphate buffer saline (PBS) at pH 7.4 (3.8–11.6% range). All compounds exhibited weak inhibition of the COX-1 isozyme (IC₅₀ = 5.8–17.0 μM range) in conjunction with weak or modest inhibition of the COX-2 isozyme (IC₅₀ = 1.6–14.4 μM range). The most potent AI agent 5-[4-(O²-ammonium diazen-1-ium-1,2-diolato)phenyl]-1-(4-sulfamoylphenyl)-3-trifluoromethyl-1H-pyrazole exhibited a potency that was about fourfold and twofold greater than that observed for the respective reference drugs aspirin and ibuprofen. These studies indicate that use of a cupferron template constitutes a plausible drug design approach targeted toward the development of AI drugs that do not cause gastric irritation, or elevate blood pressure and induce platelet aggregation that have been associated with the use of some selective COX-2 inhibitors.     

Nitric oxide donor increases the efficiency of cytostatic therapy and retards the development of drug resistance.

The potentiality to increase the chemotherapeutic effectiveness of some cytostatics in low, subtherapeutic doses in combination with nitric oxide (NO) donor has been shown. This type of combined therapy results in significant increase in life span and number of survivors among mice bearing leukemias P388 and L-1210. A similar effect was observed for intracerebral leukemia P388 transplantation. In this case the life span of mice treated with cyclophosphamide and NO donor increased by three times in comparison to therapy with cyclophosphamide alone. The coinjection of nitric oxide donor and cytostatics improved the antimetastatic activity of the cytostatics: the index of melanoma B16 metastasis inhibition at the cyclophosphamide monotherapy is 50%; on addition of NO donor the index is over 80%. Comparative studies of NO donor (organic nitrate) and a similar compound in which ONO(2) moieties were replaced by OH groups demonstrated that the presence of NO(2) is required for adjuvant activity of compounds and confirmed that nitric oxide modifies the antitumor effects of cytostatics. It is shown also that nitric oxide donor retards the development of drug resistance to cyclophosphamide.     

A novel mitochondria-localizing nitrobenzene derivative as a donor for photo-uncaging of nitric oxide

We report a novel green-fluorescent NO donor, NBDNO, bearing a 2,6-dimethylnitrobenzene moiety for photocontrollable NO release and a triphenylphosphonium moiety for targeting to mitochondria. Photorelease of NO from NBDNO was confirmed by means of ESR analysis in aqueous solution. Intracellular release of NO from NBDNO was confirmed by using DAR-4M AM, an NO-specific fluorescence probe. NBDNO was colocalized with MitoRed, a mitochondrial stain, in HCT116 colon cancer cells. Our results indicate that NBDNO is an effective NO donor for time-controlled, mitochondria-specific NO treatment.     

Human S-nitroso oxymyoglobin is a store of vasoactive nitric oxide

Nitric oxide (.NO) regulates vascular function, and myoglobin (Mb) is a heme protein present in skeletal, cardiac, and smooth muscle, where it facilitates O(2) transfer. Human ferric Mb binds .NO to yield nitrosylheme and S-nitroso (S-NO) Mb (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) J. Biol. Chem. 276, 3991-3998). Here we show that human ferrous oxy-myoglobin (oxyMb) oxidizes .NO, with a second order rate constant k = 2.8 +/- 0.1 x 10(7) M(-1).s(-1) as determined by stopped-flow spectroscopy. Mixtures containing oxyMb and S-nitrosoglutathione or S-nitrosocysteine added at 1.5-2 moles of S-nitrosothiol/mol oxyMb yielded S-NO oxyMb through trans-nitrosation equilibria as confirmed with mass spectrometry. Rate constants for the equilibrium reactions were k(forward) = 110 +/- 3 and k(reverse) = 16 +/- 3 M(-1).s(-1) for S-nitrosoglutathione and k(forward) = 293 +/- 5 and k(reverse) = 20 +/- 2 M(-1).s(-1) for S-nitrosocysteine. Incubation of S-NO oxyMb with Cu(2+) ions stimulated .NO release as measured with a .NO electrode. Similarly, Cu(2+) released .NO from Mb immunoprecipitated from cultured human vascular smooth muscle cells (VSMCs) that were pre-treated with diethylaminenonoate. No .NO release was observed from VSMCs treated with vehicle alone or immunoprecipitates obtained from porcine aortic endothelial cells with and without diethylaminenonoate treatment. Importantly, pre-constricted aortic rings relaxed in the presence of S-NO oxyMb in a cyclic GMP-dependent process. These data indicate that human oxyMb rapidly oxidizes .NO and that biologically relevant S-nitrosothiols can trans-(S)nitrosate human oxyMb. Furthermore, S-NO oxyMb can be isolated from cultured human VSMCs exposed to an exogenous .NO donor at physiologic concentration. The potential biologic implications of S-NO oxyMb acting as a source of .NO are discussed.     

Membrane penetration of nitric oxide and its donor S-nitroso-N-acetylpenicillamine: a spin-label electron paramagnetic resonance spectroscopic study

S-nitroso-N-acetylpenicillamine (SNAP) is a pharmacological agent with diverse biological effects that are mainly attributable to its favorable characteristics as a nitric oxide (NO)-evolving agent. It is found that SNAP incorporates readily into dimyristoyl phosphatidylcholine (DMPC) bilayer membranes; and an approximate penetration profile was obtained from the depth dependence of the perturbation that it exerts on spin-labeled lipid chains. The profile of SNAP locates it deep in the hydrophobic core of both fluid- and gel-phase membranes. The spin relaxation enhancement of spin-labeled phospholipids with nitroxide group located at different depths in DMPC membranes was determined for nitric oxide (NO) and molecular oxygen (O(2)), at close to atomic spatial resolution. The relaxation enhancement, which is proportional to the corresponding vertical membrane profile of the concentration-diffusion product, was measured in the gel and fluid phases of the lipid bilayer. No significant membrane penetration was observed in the gel phase for the two water-dissolved gases. In the fluid phase, the transmembrane profiles of NO and O(2) are similar and could be well described by a sigmoidal function with a maximum in the center of the bilayer, but that of NO is less steep and is shifted toward the center of the membrane, relative to that of O(2). These differences can be attributed mainly to the difference in hydrophobicity between the two gases and the presence of the donor in the NO experiments. The biological implications of the above results are discussed.     

The survival of skeletal muscle myoblasts in vitro is sensitive to a donor of nitric oxide and superoxide, SIN-1, but not to nitric oxide or peroxynitrite alone.

The survival of skeletal muscle myoblasts in culture after exposure either to a donor of NO, sodium nitroprusside (SNP), or ethanamine, 2,2'-(hydroxynitrosohydrazono)bis-(DETA NONOate), or to a donor of both NO and O(-)(2), 3-morpholinosydnonimine hydrochloride (SIN-1), was investigated. SIN-1 reduced clonogenic survival markedly but donors of NO alone did not. The injurious effect of SIN-1 was prevented by oxyhemoglobin or by uric acid but not by superoxide dismutase. The exposure of myoblasts to authentic peroxynitrite (ONOO(-)) or to DETA NONOate in the presence of an O(-)(2)-generating system did not reduce their survival. The results show that NO or ONOO(-) alone is not detrimental to myoblast survival and suggest that SIN-1 toxicity is, at least in part, mediated by H(2)O(2) in this myoblast culture system.     

Simultaneous measurement of intracellular Ca(2+) and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have been established. We are going to present a new method to measure intracellular calcium and NO simultaneously in endothelial cells. A new fluorescent dye (DAF-2) has been developed recently which binds NO resulting in an enhanced fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 simultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin induces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This new method of a simultaneous measurement of intracellular calcium and NO provides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NO-synthase.     

Membrane inlet for mass spectrometric measurement of nitric oxide

We point out the advantages of membrane inlet mass spectrometry for the measurement of nitric oxide in aqueous solution. The membrane inlet probe was a 1.0-cm segment of Silastic tubing attached to the vacuum inlet leading to the ion source. Silastic is a semipermeable silicon rubber that allows flux of uncharged substances including nitric oxide (NO). The use of such an inlet to measure NO has several advantages that we demonstrate in this report. It provides a direct, continuous, and quantitative determination of dissolved nitric oxide concentrations over long periods of real time. The use of such an inlet in our system had a response time of 5 to 7 s and a detection lower limit with the current model of 1.0 nM. This apparatus was used to measure the generation of NO from solutions of nitrite, NONOates, and nitroprusside. The usefulness of such an inlet in measuring NO in physiological systems is discussed.     

Aortic endothelial cells damaged by a nitric oxide donor and protected by flavonoids.

Cultured porcine aortic endothelial cells (PAEC) were exposed to four concentrations (0.00 mM - 5.00 mM) of 3-Morpholino-sydnonimine-hydrochloride (SIN-1, a nitric oxide donor). SIN-1 demonstrated a dose dependent cytotoxicity against PAEC as indicated by the thiobarbituric acid (TBA) assay. Morphologically and biochemically, the presence of selected flavonoids (morin, quercetin, or catechin) was shown to protect the PAEC from SIN-1 toxicity. Protection levels determined from the TBA assay were significant (p<0.05) for all flavonoids, with morin at 72+/-8%. Quercetin and catechin had comparable protective activities of 54+/-6% and 43+/-3%, respectively. This study supports the contention that SIN-1 is cytotoxic to PAEC and that antioxidants such as flavonoids may attenuate such toxicity.