Gursev S. Dhaunsi

Peroxisomal participation in the cellular response to the oxidative stress of endotoxin.

Exposure to a sublethal dose of endotoxin offers protection against subsequent oxidative stresses. The cellular mechanisms involved in generating this effect are not well understood. We evaluated the effect of endotoxin on antioxidant enzymes in liver peroxisomes. Peroxisomes have recently been shown to contain superoxide dismutase (SOD) and glutathione peroxidase (GPX) in addition to catalase. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment increased the specific activity of SOD and GPX in peroxisomes to 208% and 175% of control activity, respectively. These findings correlated with increases in peroxisomal SOD and GPX proteins observed by immunoblot. Although the quantity of catalase protein was increased when assessed by immunoblot analysis, the specific activity of catalase was decreased to 68% of control activity. Activation of catalase with ethanol only restored catalase activity to control levels suggesting that catalase had undergone irreversible inactivation. The observed increase in GPX activity may represent a compensatory mechanism triggered by accumulating H2O2. The data presented here suggest for the first time that mammalian peroxisomal antioxidant enzymes are altered during the oxidative injury of endotoxin treatment.

NADPH oxidase in human lung fibroblasts.

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91- phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.

Interleukin-1 and Nitric Oxide Increase NADPH Oxidase Activity in Human Coronary Artery Smooth Muscle Cells

Objective: Cytokines, nitric oxide (NO) and reactive oxygen species (ROS) are well known for their pathogenic effects in development of cardiovascular diseases. Interleukin-1β (IL-1β) is known to induce NO generation, however it is not well established if IL-1β or NO regulate production of ROS, such as superoxide anion. Therefore, the main objective of this study was to evaluate the effect of IL-1β or NO on enzyme activity of NADPH oxidase (NOX), a superoxide-generating system recently documented to participate in a variety of vascular functions. Methods: Human coronary artery smooth muscle cells (SMC) obtained from Clonetics were treated with IL-1β and NO donor, sodium nitroprusside (SNP), in culture. Nitrites accumulated in supernatants of SMC cultures were measured as an index of NO released following treatment with IL-1β. NOX enzyme activity was assayed using cytochrome c as the electron acceptor. Results: Treatment with IL-1β resulted in a 3-fold increase in the production of NO by SMC. Both IL-1β and SNP enhanced NOX activity, by 67 and 45%, respectively, following 24 h of treatment. Conclusion: This study suggests that NO or NO- generating cytokines might regulate the production of ROS in the cardiovascular system through modulation of superoxide-generating systems such as NOX.

Nitric oxide promotes mitogen-induced dna synthesis in human dermal fibroblasts through cGMP.

1. Nitric oxide (NO) is a free radical with multiple functions in cellular pathophysiology. Nitric oxide has been proven to play an important role in wound healing; however, the mechanisms by which NO may promote wound healing are not clearly understood. We have investigated the effect of NO on growth factor‐induced DNA synthesis in human dermal fibroblasts to suggest interactions between growth factors and NO as a possible mechanism for the role of NO in wound healing.

IMPAIRMENT OF PEROXISOMAL BETA -OXIDATION SYSTEM BY ENDOTOXIN TREATMENT

It is now clear that peroxisomes play a crucial role in many cellular processes, including the β-oxidation of very long chain fatty acids. Recently, mammalian peroxisomes have been shown to contain the antioxidant enzymes, superoxide dismutase and glutathione peroxidase, in addition to catalase. The presence of these enzymes in peroxisomes suggests that peroxisomes undergo oxidative stress in normal and disease states. As an indicator of the potential impact of an oxidative stress on peroxisomal functions, we evaluated the effect of endotoxin exposure on the β-oxidation enzyme system in rat liver. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment decreased the β-oxidation of lignoceric acid to 56% of control values (p<0.01). The specific activity of the rate limiting enzyme in the system, acyl-CoA oxidase, was decreased to 73% of control values (p<0.05). Immunoblot analysis revealed a 25% decrease in the 21KD subunit of the acyl-CoA oxidase protein. In contrast, the protein levels of the other enzymes in the pathway, trifunctional protein and 3-ketoacyl-CoA thiolase, were increased by 10 and 15%, respectively. These findings suggest that impairment of β-oxidation of lignoceric acid by endotoxin treatment is due primarily to a reduction in the activity and protein level of the key enzyme, acyl-CoA oxidase. Oxidative stresses such as endotoxin exposure may have deleterious effects on important peroxisomal functions, such as β-oxidation of very long chain fatty acids.