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Severe pain syndromes reduce quality of life in patients with inflammatory and neoplastic diseases, often because chronic opiate therapy results in reduced analgesic effectiveness, or tolerance, leading to escalating doses and distressing side effects. The mechanisms leading to tolerance are poorly understood. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice was consistently associated with the appearance of several tyrosine-nitrated proteins in the dorsal horn of the spinal cord, including the mitochondrial isoform of superoxide (O2-) dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase. Furthermore, antinociceptive tolerance was associated with increased formation of several proinflammatory cytokines, oxidative DNA damage, and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibition of NO synthesis or removal of O2- blocked these biochemical changes and inhibited the development of tolerance, pointing to peroxynitrite (ONOO-), the product of the interaction between O2- and NO, as a signaling mediator in this setting. Indeed, coadministration of morphine with the ONOO- decomposition catalyst, Fe(III) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, blocked protein nitration, attenuated the observed biochemical changes, and prevented the development of tolerance in a dose-dependent manner. Collectively, these data suggest a causal role for ONOO- in pathways culminating in antinociceptive tolerance to opiates. Peroxynitrite (ONOO-) decomposition catalysts may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-alpha and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-kappaB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IkappaB-alpha were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-alpha, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-kappaB, and this effect was associated with modest suppression of cytoplasmic IkappaB-alpha degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-alpha and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-kappaB transactivation or signaling.

Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-alpha and IL-1beta expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-alpha production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.