Nitric oxide sensing by chlorophyll a.

Nitric oxide (NO) acts as a signalling molecule that has direct and indirect regulatory roles in various functional processes in biology, though in plant kingdom its role is relatively unexplored. One reason for this is the fact that sensing of NO is always challenging. There are very few probes that can classify the different NO species. The present paper proposes a simple but straightforward way for sensing different NO species using chlorophyll, the source of inspiration being hemoglobin that serves as NO sink in mammalian systems. The proposed method is able to classify NO from DETA-NONOate or (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2-diolate, nitrite, nitrate and S-nitrosothiol or SNO. This discrimination is carried out by chlorophyll a (chl a) at nano molar (nM) order of sensitivity and at 293 K-310 K. Molecular docking reveals the differential binding effects of NO and SNO with chlorophyll, the predicted binding affinity matching with the experimental observation. Additional experiments with a diverse range of cyanobacteria reveal that apart from the spectroscopic approach the proposed sensing module can be used in microscopic inspection of NO species. Binding of NO is sensitive to temperature and static magnetic field. This provides additional support for the involvement of the porphyrin ring structures to the NO sensing process. This also, broadens the scope of the sensing methods as hinted in the text.

Enzymatic Sensor Detects Some Forms of Nitric Oxide Donors Undetectable by Other Methods in Living Tissues.

Studies with the use of highly sensitive enzymatic sensor have shown the presence of various forms of nitrosyl iron complexes, including those undetectable by other methods, in living tissues. All these complexes are long-living compounds and constitute the major part of nitroso compounds in the blood, muscles, liquor, and amniotic fluid.

Can summary nitrite+nitrate content serve as an indicator of NO synthesis intensity in body tissues?

Studies with the use of a highly specific enzymatic sensor demonstrated that, contrary to the common opinion, normally nitrate is in fact not present in the most important physiological fluids. NO metabolites in the amniotic fluid and semen are mainly presented by NO donor compounds. Therefore, the intensity of NO synthesis can be evaluated by the total content of all its metabolites, but not by the widely used summary nitrite+nitrate content.

Identification of circulatory and excretory metabolites of a novel nitric oxide donor ZJM-289 in rat plasma, bile, urine and faeces by liquid chromatography-tandem mass spectrometry.

ZJM-289, [2-(1-diethylaminoacetoxy)pentyl] benzoic acid-{2-methoxy-4-[2-(4-nitrooxybutoxy carbonyl)-vinyl]}phenyl ester hydrochloride, is a novel nitric oxide-donating derivative of 3-n-butylphthalide synthesised on the hypothesis that it may be hydrolysed in vivo into 3-n-butylphthalide, ferulic acid and nitric oxide in hope that the three components may exert effects on the platelets as well as on central nervous system synergistically. In this study, ZJM-289 was extensively metabolised in rats. Eight major metabolites were identified by liquid chromatography (LC)-mass spectrometry (MS)/MS in rat plasma, bile, urine and faeces after intravenous administration. Metabolites M1, M2, M3, M4 and M5 were hydrolytic products of ZJM-289, M6 and M7 was a hydroxylation product of M5, and M8 was a glucuronide of M1. The pharmacologically active metabolite ferulic acid (M3) was a major metabolite in all the biological matrixes examined. 3-n-Butylphthalide was also present at a moderate level in the circulation. And along with the previous research, the anti-platelet activity of ZJM-289 was more potent than that of 3-n-butylphthalide both in vivo and in vitro. All these findings validated the theory of drug design.

The identification of a nitrosated prodrug of the PDE-5 inhibitor aildenafil in a dietary supplement: a Viagra with a pop.

A new unapproved analogue of sildenafil was detected in capsules of a herbal dietary supplement promoted as a libido enhancing product. Using LC-DAD-MS, MS-MS, HRMS, IR and NMR the analogue was shown to be a derivative of the PDE-5 inhibitor aildenafil with a nitrosamine moiety. A hydrolysis experiment showed that the new analogue was a prodrug of aildenafil and was therefore named nitroso-prodenafil. A capsule contained 108 mg of nitroso-prodenafil which is equivalent to 84 mg of aildenafil and 5.1 mg of nitrogen monoxide (NO). Although it is unknown how much NO can be usefully generated there is 3-fold more NO present than in a 10 mg isorbide nitrate tablet. Both PDE-5 inhibitors and nitrosamines cause vasodilatation by increasing levels of NO. To their coincidental use is warned against because it may cause a fatal drop in blood pressure. In addition, nitrosamines are known carcinogens. This is the first time a PDE-5 inhibitor and a potential NO donor were identified in one molecule. The findings indicate the dangerous level of advancement in medicinal chemistry by producers of unapproved drugs.

A quantitative approach to nitric oxide inhibition of terminal oxidases of the respiratory chain.

Inhibition of terminal respiratory oxidases by nitric oxide (NO) plays important physiological roles in signaling and host defense. Using a bacterial quinol oxidase and mitochondrial cytochrome c oxidase, this chapter describes simple polarographic methods to quantify the kinetic characteristics of inhibition by NO. This chapter points out the inherent pitfalls of both experimental design and data analysis and compares alternative methods. Additionally, it describes a system designed to acquire polarographic and spectral data simultaneously to permit identification of spectral intermediates under defined conditions.

Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources.

Because of controversies in the literature on nitric oxide (NO) production by plants, NO detection by the frequently used diaminofluorescein (DAF-2 and DAF-2DA) and by chemiluminescence were compared using the following systems of increasing complexity: (i) dissolved NO gas; (ii) the NO donor sodium nitroprusside (SNP); (iii) purified nitrate reductase (NR); and (iv) tobacco cell suspensions. Low (physiological) concentrations (< or =1 nM) of dissolved NO could be precisely quantified by chemiluminescence, but caused no DAF-2 fluorescence. In contrast to NO gas, SNP, NR, or cell suspensions produced both good DAF fluorescence and chemiluminescence signals which were completely (chemiluminescence) or partly (DAF fluorescence) prevented by NO scavengers. Signal strength ratios between the two methods were variable depending on the NO source, and eventually reflect variable NO oxidation. DAF fluorescence in cell suspension cultures was also increased by an as yet unidentified compound(s) released from cells into the medium. These compounds gave no chemiluminescence signal and were not produced by NR-free mutants. Their production was stimulated by anoxia, by inhibitors of mitochondrial electron transport, and by the fungal elicitor cryptogein. Thus, changes in DAF fluorescence are not necessarily indicative for NO production, but may also reflect NO oxidation and/or production of other DAF-reactive compounds.

Injectable formulation of disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), an ultrafast nitric oxide donor prodrug.

PROLI/NO is an agent of structure XN(O)==NONa (X = L-prolyl) whose 2-s half-life for nitric oxide (NO) release at physiological pH makes it an excellent prodrug for localizing NO's therapeutic effects at the site of application, but a difficult one to formulate and certify as pure. Despite its extraordinary thermal and hydrolytic instability, however, PROLI/NO could be formulated as an injectable drug by dissolving it in cold 0.1 M sodium hydroxide containing 5% D-mannitol, then quickly ultrafiltering and lyophilizing it in evacuated septum vials. No evidence for decomposition was seen in the contents of these evacuated vials when stored at -20 degrees C over a 140-day observation period, as judged by quantifying NO release in simulated infusate solutions (10 mM carbonate/bicarbonate, pH 10.5). The only hydrolysis products detected were NO, nitrite ion, proline, and N-nitrosoproline, all products of normal human physiological processes.

The effects of acute and chronic radiation on endothelium- and no-dependent vascular reactivity.

Endothelium- and NO-dependent relaxation of aortic segments and basal coronary flow and response of coronary vessels to exogenous NO in the Langendorff-perfused rat hearts were examined on the 3rd, 10th, 30th and 90th days following whole body irradiation in 1 Gy dose with different dose rate. NO-mediated elevation of coronary flow and increased aortic endothelium-dependent vasodilation were found at the early stage after acute irradiation (137Cs, 9 x 10(-4) Gy/s), while vascular reactivity to exogenous NO was not changed. Chronic irradiation (137Cs, 2.3 x 10(-7) Gy/s) significantly impaired endothelium-dependent relaxation within whole experimental period and decreased NO component of basal coronary flow at the 3rd, 10th and 90th day. It was associated with attenuated reactivity to NO-donor in aorta one month after irradiation and in the coronary vessels--shortly after irradiation. In the delayed period endothelial dysfunction and the diminution of NO-dependent coronary flow were mediated by selective impairment of NO synthesis/release. Decreased survival rate of the lethally-irradiated Nwnitro-L-arginine methyl ester-treated animals revealed radiosensitizing properties of NO synthase inhibition. The data obtained indicate the involvement of NO in the alterations of vascular reactivity depending on the dose rate and on the interval after the irradiation.

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release.

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.