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Highlights: [•] In ischemia copper dissociates from its carrier proteins, i.e., albumin. [•] Free Cu2+ ions are reduced to Cu1+ by ascorbate, Fe2+ and/or GSH. [•] Copper-catalyzed NO formation from nitrite exerts biological activity. [•] Cu1+-catalyzed NO formation represents a new protective principle to ischemic stress. [Copyright &y& Elsevier]

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 Cu2+ ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu2+ ions to Cu1+ 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. [Copyright &y& Elsevier]

Abstract: Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively. [Copyright &y& Elsevier]