Differential Role of Extra- and Intracellular Superoxide Anions for Nitric Oxide-mediated Apoptosis Induction

Nitric oxide (NO) has recently been shown to mediate apoptosis induction selectively in transformed fibroblasts, in contrast to their nontransformed parental cells. Here we show that NO-mediated apoptosis induction in transformed fibroblasts can be divided into two major phases. During phase 1, peroxynitrite is generated by the interaction of extracellular superoxide anions with NO and the intracellular glutathione level is subsequently lowered. This defines the beginning of phase 2, in which NO-mediated signaling depends on intracellular superoxide anions exclusively. The resultant peroxynitrite seems to activate the mitochondrial permeability transition pore and thus triggers execution of apoptosis. Experimental depletion of intracellular glutathione causes a drastic decrease in the length of phase 1 in transformed cells and renders nontransformed cells sensitive to NO-mediated apoptosis induction. These findings allow the prediction that either induction of superoxide anion generation or glutathione depletion may render cells sensitive to NO-mediated apoptosis induction. Nitric oxide (NO) is a free radical with diverse biological functions of central importance (1, for review see ref. 2). It arises from the guanodino group of L-arginine in a NADPH- dependent reaction catalyzed by constitutively expressed or inducible NO synthases (NOS). NO is able to pass cellular membranes (3), to decrease the intracellular glutathione pool (4), to regulate gene expression via interaction with the zinc finger transcription factor SP1 (5) and to up-regulate p53 gene expression (6). The neurotrophic function of NO switches to cell death induction in midbrain cultures after glutathione depletion (7). Direct apoptosis induction by NO has been reported for