Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Function and design of the Nox1 system in vascular smooth muscle cells.

BACKGROUND: Recent studies have demonstrated that the activation of NADPH oxidase 1 (Nox1) plays an important role in the control of reactive oxygen species and their involvement in vascular physiology and pathophysiology. In order to function properly, Nox1 needs to be available in an optimal state, where it is ready to respond appropriately and efficiently to upstream signals. It must also be able to return quickly to this state as soon as the input signal disappears. While Nox1 activation has been discussed extensively in recent years, mechanisms for enzyme disassembly and proper subunit recovery have not received the same attention and therefore require investigation. RESULTS: We study the Nox1 system in vascular smooth smucle cells and propose four potential disassembly mechanisms. The analysis consists primarily of large-scale Monte-Carlo simulations whose results are essentially independent of specific parameter values. The computational analysis shows that a specific profile of subunit concentrations is crucial for optimal functioning and responsiveness of the system to input signals. Specifically, free p47(phox) and inactive Rac1 should be dominant under unstimulated resting conditions, and the proteolytic disassembly pathway should have a low flux, as it is relatively inefficient. The computational results also reveal that the optimal design of the three subunit recovery pathways depends on the intracellular settings of the pathway and that the response speeds of key reversible reactions within the pathway are of great importance. CONCLUSIONS: Our results provide a systematic basis for understanding the dynamics of Nox1 and yield novel insights into its crucially important disassembly mechanisms. The rigorous comparisons of the relative importance of four potential disassembly pathways demonstrate that disassembly via proteolysis is the least effective mechanism. The relative significance of the other three recovery pathways varies among different scenarios. It is greatly affected by the required response speed of the system and depends critically on appropriate flux balances between forward and reverse reactions. Our findings are predictive and pose novel hypotheses that should be validated with future experiments.