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Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.

These experiments examined the metabolic properties of the canine respiratory muscles. Because the costal diaphragm (COD), crural diaphragm (CRD), parasternal intercostals (PI), triangularis sterni (TS), and transversus abdominis (TA) are active during quite breathing in the dog, we hypothesized that these muscles would have different metabolic profiles (i.e., higher oxidative and antioxidant enzyme activities) compared with ventilatory muscles recruited only at increased ventilatory requirements [e.g., scalene (SC) and external oblique (EO)] and locomotor muscles [e.g., deltoid (DEL)]. To test this hypothesis, muscle samples were removed from six healthy adult dogs and analyzed to determine the activities of citrate synthase (CS), phosphofructokinase (PFK), 3-hydroxyacyl-CoA dehydrogenase (HADH), and superoxide dismutase (SOD). The activities of these enzymes were interpreted as relative measures of metabolic capacities, and enzyme activity ratios were considered as representing relationships between different metabolic pathways. Analysis revealed that CS and HADH activities were significantly higher (P < 0.05) in the PI, COD, CRD, and TS compared with those in all other muscles. Muscles with the lowest CS, HADH, and SOD activities (i.e., SC, TA, EO, DEL) generally had the highest PFK activities, Furthermore, the PFK/CS ratio was significantly lower in the PI, COD, CRD, and TS compared with that in all other muscles studied. These data support the notion that the canine PI, COD, CRD, and TS are metabolically different from other key ventilatory muscles.