Pro- and Anti-Apoptotic Effects of Nitric Oxide in Irradiated Keratinocytes: The Role of Superoxide

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Abstract
Ultraviolet radiation (UV) induces apoptosis in keratinocytes by both p53- and death receptor-dependent pathways. It also generates free radicals in keratinocytes, including the synthesis of nitric oxide (NO) by constitutive and inducible NO synthases (NOS). NO has both pro- and anti-apoptotic effects. We wished to determine which of these was predominant in keratinocytes. Human CCD1106 keratinocytes were irradiated with UVB in the presence and absence of several NOS antagonists. Apoptosis was measured by flow cytometry with annexin V binding. NOS antagonism consistently altered UVB-induced apoptosis measured 18 h after irradiation. In 9 of 13 experiments, NOS antagonism increased apoptosis. However, in 4 of 13 experiments, NOS antagonism reduced apoptosis. We postulated that the variable effects of NO might be due to a critical balance between UVB-induced NO and superoxide production. We predicted that NO would be anti-apoptotic in the presence of low O–2, but pro-apoptotic when NO combined with O–2 to form peroxynitrite. Though superoxide dismutase reduced apoptosis after UVB, addition of peroxynitrite did not affect apoptosis. We conclude that NO released by UV irradiation is anti-apoptotic; however, the levels of O–2 may be a determinant of NO action.