It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.

Tumor necrosis factor plays a critical role in airway smooth muscle hyperresponsiveness observed in asthma. However, the mechanisms underlying this phenomenon are poorly understood. We investigated if tumor necrosis factor-stimulated airway smooth muscle produced reactive oxygen species, leading to muscular hyperresponsiveness. Tumor necrosis factor increased intracellular and extracellular oxidants production in guinea pig airway smooth muscle cells and tissue homogenates. This production was abolished by inhibitors of NADPH oxidase (diphenylene iodinium or apocynin) and was enhanced by NADPH, whereas inhibitors of mitochondrial respiratory chain, nitric-oxide synthase, cyclooxygenase, and xanthine oxidase had no effect. NADPH oxidase subunits p22 Collectively, these results demonstrate that tumor necrosis factor-stimulated airway smooth muscle produces oxidants through a NADPH oxidase-like system, which plays a pivotal role in muscle hyperresponsiveness and myosin light chain phosphorylation.Asthma is a complex inflammatory disease of the lung whose incidence, morbidity, and mortality have dramatically increased worldwide over the last two decades. Airway inflammation and ASM 1 hyperresponsiveness, leading to an increased airway resistance, are characteristic features of asthma (1). The inflammatory response in the asthmatic lung is characterized by an infiltration of the airway wall by mast cells, lymphocytes, and eosinophils. Activation of these cells results in the release of a plethora of inflammatory mediators that individually or in concert induce changes in the airway wall geometry and produce the symptoms of the disease. There is increasing evidence that one of these mediators, the pro-inflammatory cytokine TNF may be one of the primary components responsible for the ASM hyperresponsiveness observed in asthma (see Ref. 2 for review). However, the mechanism of this TNF-induced ASM hyperresponsiveness remains unclear. TNF may act indirectly, via the release of other inflammatory or bronchoconstricting agents such as leukotrienes, by inflammatory cells (2), or directly on ASM cells that express TNF receptors (3). Indeed, different investigators have shown that a short time incubation of tracheal smooth muscle strips with TNF enhances the contractile response to acethylcholine (4, 5) secondary, at least partially, to an increase in MLC phosphorylation (6).This direct effect of TNF on ASM contractility could be mediated by ROS synthesized by the muscular cells. At least three lines of evidence support this hypothesis: 1) TNF leads to the generation of ROS in various cell systems (7,8), 2) incubation of guinea pig tracheal smooth muscle with SOD decreases the contractile response to metacholine (9), suggesting that endogenous ROS can increase ASM contractility, and 3) ROS could increase phosphorylation of the MLC by activating the MLC kinase and/or by inhibiting the MLC phosphatase, as previously described with other kinases/phosphatases systems (10). However, very few studies investigated the capacity of AS...

Pharmacological evidence supports a role of a transient decreased endogenous nitric oxide (NO) synthesis in ovalbumin (OVA)-induced early airway hyperresponsiveness in guinea pigs. However, no data are available regarding the expression and activity of the constitutive NO synthases (cNOS; NOS1 and NOS3, nNOS and eNOS, respectively) in this model. Therefore, we evaluated cNOS activity (conversion of L-[3H]arginine to L-[3H]citrulline in the presence of Ca2+ and calmodulin), nitrate and nitrite (NOx) concentration (modified Griess method), and NOS1 and NOS3 protein expression (Western blot) in lung homogenates and in the tracheal smooth muscle from OVA-immunized and multiple aerosol-challenged guinea pigs (six challenges, once daily). The expression and activity of the inducible NOS isoform (NOS2), the levels of exhaled NO, and the in vivo airway reactivity were also determined. Constitutive NOS activity and NO(x) concentration were significantly lower 6 h after the last OVA challenge as compared with saline exposure, being similar at 24 h. Expression of NOS1 paralleled cNOS activity, which was reduced 6, but not 24 h after OVA challenge. The decrease in NOS1 expression was accompanied by a significant decrease in the amounts of exhaled NO and by a maximal airway hyperresponsiveness to histamine. The levels of NOS3 were not modified at the two time points evaluated, and no NOS2 expression and activity were found at any time point. Similar modifications were observed in the tracheal smooth muscle. We conclude that OVA stimulation in immunized guinea pigs induced a transient reduction in NOS1 protein expression and activity in the respiratory system, which probably participates in airway hyperresponsiveness.