Ultraviolet B Light-induced Nitric Oxide/Peroxynitrite Imbalance in Keratinocytes-Implications for Apoptosis and Necrosis

Elevation of nitric oxide (NO center dot) can either promote or inhibit ultraviolet B light (UVB)-induced apoptosis. In this study, we determined real-time concentration of NO center dot and peroxynitrite (ONOO-) and their role in regulation of membrane integrity and apoptosis. Nanosensors (diameter 300-500 nm) were used for direct in situ simultaneous measurements of NO center dot and ONOO- generated by UVB in cultured keratinocytes and mice epidermis. An exposure of keratinocytes to UVB immediately generated ONOO- at maximal concentration of 190 nm followed by NO center dot release with a maximal concentration of 91 nm. The kinetics of UVB-induced NO center dot/ONOO- was in contrast to cNOS agonist stimulated NO center dot/ONOO- from keratinocytes. After stimulating cNOS by calcium ionophore (CaI), NO center dot release from keratinocytes was followed by ONOO- production. The [NO center dot] to [ONOO-] ratio generated by UVB decreased below 0.5 indicating a serious imbalance between cytoprotective NO center dot and cytotoxic ONOO--a main component of nitroxidative stress. The NO center dot/ONOO- imbalance increased membrane damage and cell apoptosis was partially reversed in the presence of free radical scavenger. The results suggest that UVB-induced and cNOS-produced NO center dot is rapidly scavenged by photolytically and enzymatically generated superoxide (O(2)center dot-) to produce high levels of ONOO-, which enhances oxidative injury and apoptosis of the irradiated cells.