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[Nitrogen monoxide (NO)--the active principle of organic nitrates].

Ableitung Pharmakologie, Schwarz Pharma AG, Monheim, Deutschland.
Schweizerische Rundschau fur Medizin Praxis = Revue suisse de medecine Praxis 11/1993; 82(42):1167-71.
Source: PubMed

ABSTRACT Although organic nitrates have been used in the treatment of patients with angina pectoris for more than 100 years, their mechanism of action was only disclosed during the last years. In the seventies it became already clear that nitrates act via the intracellular messenger cyclic 3,5'-guanosine-monophosphate (cGMP). Later on, S-nitrosothiols were suggested as possible intermediary messengers arising during metabolism of the nitrates. In parallel with the discovery of the endothelium-derived relaxing factor (EDRF) and its biochemical identification as nitric oxide (NO), it became clear that organic nitrates act via the release of NO in the vascular wall and thus by using metabolic pathways identical to those of endogenous EDRF. The target-enzyme for nitrates or for the NO released by them, respectively, thus is the soluble guanylylcyclase. The rate of enzymic stimulation induced by a given nitrate correlates closely with the rate of measured NO production from the nitrate molecule. The highest NO production was detected with nitroglycerin, followed by the group of dinitrates and mononitrates. In the vessel wall both endothelial cells as well as vascular smooth muscle cells can transform nitrates into NO. This might explain, why the antiaggregatory effect of nitrates is more pronounced in the presence of these cells or in vivo than it is in the absence of vascular cells in vitro. In spite of some differences in metabolism, nitrates are thus closely related by their end-product to the endothelium-derived relaxing factor and represent, therefore, an adequate substitution for NO missing in diseased blood vessels.

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Available from: Martin Feelisch, May 10, 2014
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    ABSTRACT: Topical application of nitric oxide (NO) has been shown to exert beneficial effects in the therapy of chronic wounds, impaired microcirculation, and skin infections. Nitrite acidified by ascorbic acid has been widely used in many studies as NO-donor system, unfortunately with inflammatory and toxic effects on the treated skin due to unregulated excessive NO generation, low pH and possible toxic side products. Here we describe an essentially modified nitrite based NO generating system that avoid the mentioned unwanted side effects on human skin by using a pH-stable acetate/acetic acid buffer with a skin neutral pH of 5.5 and sodium ascorbate. In order to overcome the shortcoming of lower NO yields due to the higher pH-value and low nitrite concentrations, we have determined additionally the influence of copper ions. To investigate the influence of different NO release and penetration kinetics on NO-induced toxicity, we have developed a fibroblast assay using cell culture plates with gas permeable bottoms. The results show clearly that the donor system can achieve a sustained NO generation without generating high peaks. Furthermore, the presence of Cu(2+) ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu(2+) ions to Cu(1+) by ascorbate. Finally, we have found that apart from the NO dose the NO release kinetics had a significant influence of cell toxicity. Thus, application of comparable NO amounts within a time interval of 600s led to the development of variable cell toxicities, which predominantly depended on the NO concentration values generated in the first 200s. In summary, we here describe a novel nitrite-based NO-donor system that can provide well defined NO concentrations at skin neutral pH-values for side effect poor topical dermal application, i.e. in the therapy of chronic wounds and impaired microcirculation.
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    ABSTRACT: Stickstoffmonoxid (NO•) spielt in der Hautphysiologie eine wichtige Rolle und ist an vielen biologischen Prozessen, wie z.B. Hautbräunung, Wundheilung sowie Wachstum und Differenzierung von Keratinozyten und Fibroblasten beteiligt. Des Weiteren ist NO• ein zellprotektives Agens, welches z.B. effektiv vor der UVA-induzierten Apoptose schützen kann. NO• wird in vivo enzymatisch durch NO-Synthasen gebildet, kann aber auch enzymunabhängig durch UV- oder pH-induzierten Zerfall natürlich vorkommender NO-Derivate (NOD), wie Nitrit und S-Nitrosoverbindungen, in hohen Mengen entstehen. Der Mechanismus und die Rolle der nicht-enzymatischen NO-Bildung in der menschlichen Haut sind weitgehend unbekannt. In Rahmen dieser Arbeit konnten wir erstmals zeigen, dass UVA-Strahlung unter physiologischen Bedingungen das Nitrit-Anion unter der Generierung von NO• effektiv spalten kann. Dabei entstehen redoxaktive Zwischenprodukte die mit dem entstehenden NO reagieren können und dadurch die NO-Generierung limitieren. Anderseits werden durch die Verwendung starker Antioxidanzien diese Zwischenprodukte eliminiert und führen so zu einer maximalen Ausbeute der NO-Freisetzung. In einen weiteren Ansatz wurde überprüft, ob die nicht-enzymatische NO-Generierung aus Nitrit als protektives NO-Donorsystem in einem Modell der UVA-induzierten Toxizität humaner Fibroblasten Verwendung finden kann. Entgegen unserer Erfahrungen mit Zellen der Ratte, konnten wir mit humanen Hautfibroblasten bereits bei supraphysiologischen Nitritkonzentrationen von 100 µM eine signifikante Zunahme der UVA-induzierten Zellschäden beobachten. Als das eigentliche zelltoxische Agens dieser Reaktion konnten wir das Stickstoffdioxidradikal (NO2•) identifizieren. Eine Veränderung des im System entstandenen NO•/ NO2•-Verhältnisses, z.B. durch die exogene Zugabe von NO oder eine NO2•-Eliminierung durch Vitamin C, führte hingegen zu einem Schutz der Zellen vor UVA-induzierten Zelltod. Somit konnten wir erstmal zeigen, dass der UVA-bedingte Nitritzerfall als ein Schutzsystem verwendet werden kann, wenn es gelingt die Bildung der toxischen Zwischenprodukte zu verhindern oder diese zu eliminieren. Interessanterweise führten Glutathion und Trolox, zwei potente NO2•-Scavenger, zu einer Steigerung der UVA/Nitrit-induzierten Toxizität. Diese paradoxen Befunde konnten wir auf die Generierung toxischer Glutathionyl- bzw. Phenoxyl-Radikalen zurückführen. Unsere Ergebnisse zeigen somit imposant, dass in redoxabhängigen Systemen vermeintlich positiv wirksame Agenzien toxisch wirken können. Bei der Charakterisierung der physiologischen Rolle von natürlicherweise, intrazellulärer vorkommenden NO-Derivaten konnten wir mittels Laser-Scanning-Mikroskopie erstmals nachweisen, dass in der Zelle hauptsächlich Nitrit unter Einfluss von UVA-Strahlung zerfällt und NO generiert. Fibroblastenkulturen, in denen experimentell eine signifikante Verarmung des intrazellulären Nitrits erreicht werden konnte, wiesen eine fehlenden UVA-induzierte intrazelluläre NO-Generierung und eine gleichzeitige signifkante Erhöhung der Empfindlichkeit der Zellen gegenüber UVA-induzierten Zellschäden auf. Die Substitution der nitritverarmter Zellen mit physiologischen Nitritkonzentrationen (10 µM) konnte hingegen die gesehene erhöhte Empfindlichkeit gegenüber UVA-Strahlung aufheben. Somit konnten wir hier erstmals beweisen, dass intrazelluläre, photoaktive NO-Derivate, wie Nitrit, eine physiologische Rolle im Rahmen des Zellschutzes vor UVA-Strahlung erfüllen und die nicht-enzymatische NO-Bildung ein schnell agierendes, natürliches Schutzprinzip der menschlichen Haut darstellt. Nitric oxide (NO•) plays a pivotal role in the physiology of the human skin and is involved in many biological processes like tanning, wound healing as well as the proliferation and differentiation of keratinocytes and fibroblasts. Furthermore NO• can act as a cell protective agent and prevent effectively e.g. from UVA-induced apoptosis. NO• can be produced in vivo enzymatically by NO-synthases. Additionally NO can generated in high quantities by enzyme independently UV- and pH-induced decomposition of naturally occurring NO-derivates (NOD) like nitrite and nitroso-compounds. Function and mechanism of non-enzymatic NO-generation in human skin is largely unknown. Within this work we could show for the first time that under physiological conditions UVA-radiation can decompose effectively nitrite anions, which result in NO-formation. During nitrit decomposition occur reactive intermediates which react with NO and decrease that way the NO-generation. But an elimination of this intermediates by strong antioxidants leads to a maximal yield of NO. In a further approach we examined the optional use of the non-enzymatic NO-generation from nitrite as a NO-donor system again UVA-induced cell damage in human skin fibroblasts. Contrary to our experience with rat cells supraphysiological concentrations of nitrite up 100 mM already leaded to a significant increase of UVA-induced cell damage. We could identify NO2• as the toxic component. Alteration of the NO•/ NO2• ratio by exogenous addition of NO or NO2•-Scavenger like ascorbic acid, however, leaded to a protection from the sawn UVA/nitrite-induced cell death. Hence we could demonstrate for the first time that the UVA-induced decomposition of nitrite can be used as a cell protection system, if the resulting toxic intermediates can be prevented or eliminated. Interestingly glutathione and trolox, two potent NO2•-scavengers, caused an increase of the UVA/nitrite-induced toxicity. The UVA-induced generation of glutathionyl-radicals by glutathion and phenoxyl-radicals by trolox respectively in the presence of nitrite can explain these paradoxical results. Thus, our results show impressively that supposed protective substances can become toxic in redox-depending systems. In a further characterisation of the physiological role of naturally intracellular occurring NOD, we could show the first time by using Laserscanning-Microskop-Technique, that UVA-radiation can generate intracellulary NO by decomposition of nitrite. Fibroblasts cultures which nitrite-amount had been reduced experimentally, revealed a lower UVA-induced intracellular NO-formation accompanied by a significant higher susceptibility to UVA-induced cell damage. Substitution of the nitrite-depleted cells with physiological concentration of nitrite (10 µM), however, could restore this higher susceptibility. So we demonstrate for the first time that intracellular, photolabiles NO-derivates like nitrite take place in cell protection against UVA-radiation and that non-enzymatic NO-generation by nitrite-decomposition is a naturally rapidly acting principle in the human skin.