| Excessive production of the free radical nitric oxide (NO) by NO synthases is implicated in pathological disorders, including neurodegenerative diseases, diabetes, and chronic inflammatory diseases. The function of NO in cellular signaling and cell death depends on the concentration of NO and the environment in which NO is produced. Immune cells produce NO at high nanomolar to low micromolar concentrations (in combination with reactive oxygen species) to kill target cells. This oxidative burst is toxic to surrounding cells or invading pathogens by way of DNA, protein, and lipid damage. In contrast, lower NO concentrations can protect cells from death by a variety of mechanisms, which may even protect against otherwise toxic NO doses, termed "adaptive NO resistance".; One of the main goals of this work was to define the pathways involved in NO-induced cell death (Chapters 2 and 3). NO-mediated cell death was found to have both apoptotic and necrotic characteristics in a variety of primary and immortalized fibroblast cell lines. NO-induced apoptosis proceeds through a p53-dependent mechanism, but p53-independent cell death occurred in fibroblast and lymphoblastoid cells. NO also participates in a "feed-forward" mechanism of cytotoxicity by inducing an iNOS-associated activity that was required for NO-induced cellular damage and cell death.; Another goal of this project was to determine possible mechanisms of adaptive NO resistance (Chapter 4). The primary human lung fibroblasts, IMR-90, were significantly protected against a toxic dose of NO when pretreated with a 10-fold lower level NO for 16 hours. This adaptive NO resistance required transcription but did not depend on heme oxygenase activity (shown to be important for NO resistance in neuronal cells). Adaptive NO resistance suppressed markers of apoptotic cell death, but low level NO pretreatment actually potentiated p53 activation. Treatment with low level NO induced several possible protective mechanisms, including induction of p21WAF1/CIP1, repression of NO-induced iNOS activity, and induction of signaling dependent on cyclic adenosine monophosphate (cAMP)/cAMP response element binding protein (CREB). These results provide insight into the mechanisms of NO-induced cytotoxicity and shed light on cellular adaptive resistance mechanisms utilized to avoid such cytotoxicity. |
