Biologische Aspekte der nicht-enzymatischen Stickstoffmonoxid-Generierung durch UVA-induzierten Nitritzerfall in humanen Hautfibroblasten

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