Reactive oxygen intermediates modulate skeletal muscle contraction, but little is known about the role of nitric oxide (NO). Here we show that rat skeletal muscle expresses neuronal-type NO synthase and that activity varies among several respiratory and limb muscles. Immunohistochemistry showed prominent staining of type II (fast) fibre cell membranes with antibodies against neuronal-type NO synthase. NO synthase activity in muscles correlated with type II fibre density. Resting diaphragm muscle produced detectable NO chi, but no reactive oxygen intermediates. In contrast, actively contracting muscle generated increased levels of reactive oxygen intermediates. Contractile function was augmented by blockers of NO synthase, extracellular NO chelation, and guanylyl cyclase inhibition; it was depressed by NO donors and by increased levels of cyclic GMP. Force-frequency plots of different muscles showed an inverse correlation between NO synthase activity and force development. Our results support two physiological functions of NO in skeletal muscle. The first is to promote relaxation through the cGMP pathway. The second is to modulate increases in contraction that are dependent on reactive oxygen intermediates and which are thought to occur through reactions with regulatory thiols on the sarcoplasmic reticulum.
In this issue of TheJournal Clerch et al. (1) show that hyperoxia regulates lung manganese superoxide dismutase (MnSOD) through a nonreceptor-mediated pathway involving G proteins. These observations suggest a number of possibilities regarding the cell involved and signaling mechanism used. Regu lation of MnSOD expression is a critical element in the lung's response to multiple forms of oxidant stress. It is likely that the location of MnSOD in mitochondria imparts protection to electron transport chain components enabling maintenance of cell energy sources under conditions of metabolic stress. Exposure to hyperoxia leads to a specific upregulation ofMnSOD in the mitochondria of alveolar epithelial type II cells (2). Selective over-expression of MnSOD in the mitochondria oftype II cells protects mice against hyperoxic stress (3). Indeed, the enhanced whole lung expression of MnSOD identified by Clerch et al. (1) is likely to have occurred in type II cells. These cells have a number of specialized functions designed to protect the host against inflammatory stimuli, microbes, and pollutants. In particular, they express extracellular (EC)-SOD and produce the bioactive radicals superoxide (02-) and nitric oxide (NO) (4). Selected biological functions of these radicals may derive from their rapid interaction to form peroxynitrite (OONO-). Which species predominates at the cell surface will depend on the rates and sites ofproduction ofthe primary radicals and the local concentrations of antioxidant enzymes such as EC-SOD. In this context one wonders if EC-SOD is not also regulated in the model of Clerch et al. (1). Two important questions are raised by this study. First, what is the molecular mechanism by which small diffusible ligands are recognized (i.e., what is the molecular sensor)? Second , how are these redox signals transduced into changes in gene expression? It has become increasingly clear that redox-active species such as O2 and NO-play important servoregu-latory roles through activation of cytosolic enzymes and transcription factors. Signaling by these redox species may be initiated in or at the plasma membrane (1, 5). Indeed, NO and H202 have been shown to activate G proteins (6). A distinctive feature shared by redox-active biomolecules is that they exert biological activity by virtue of their chemical reactivity, as opposed to the traditional noncovalent interactions of ligands with receptors. Metal-or sulfur-containing proteins are molecular targets for these diffusible signals. Critical thiols on the G protein are, therefore, candidate regulatory sites. Such activation of G proteins by S-nitrosylation (7) would be consistent with reports that NO. opposes pertussis toxin-mediated ADP-ribosylation ofcysteinyl residues (6). Analogous covalent interactions of O2T with protein thiols should be entertained. Con-formational changes induced in the protein likely serve as a switching mechanism to transduce the chemical signal into a physiological response. It is possible that redox-active biomolecules (O2 and ...
EC activation and dysfunction have been linked to a variety of vascular inflammatory disease states. The function of microRNAs (miRNAs) in vascular EC activation and inflammation remains poorly understood. Herein, we report that microRNA-181b (miR-181b) serves as a potent regulator of downstream NF-κB signaling in the vascular endothelium by targeting importin-α3, a protein that is required for nuclear translocation of NF-κB. Overexpression of miR-181b inhibited importin-α3 expression and an enriched set of NF-κB-responsive genes such as adhesion molecules VCAM-1 and E-selectin in ECs in vitro and in vivo. In addition, treatment of mice with proinflammatory stimuli reduced miR-181b expression. Rescue of miR-181b levels by systemic administration of miR-181b "mimics" reduced downstream NF-κB signaling and leukocyte influx in the vascular endothelium and decreased lung injury and mortality in endotoxemic mice. In contrast, miR-181b inhibition exacerbated endotoxin-induced NF-κB activity, leukocyte influx, and lung injury. Finally, we observed that critically ill patients with sepsis had reduced levels of miR-181b compared with control intensive care unit (ICU) subjects. Collectively, these findings demonstrate that miR-181b regulates NF-κB-mediated EC activation and vascular inflammation in response to proinflammatory stimuli and that rescue of miR-181b expression could provide a new target for antiinflammatory therapy and critical illness.
Idiopathic pneumonia syndrome (IPS) refers to diffuse, non-infectious pneumonia that occurs after allogeneic bone marrow transplantation (BMT). We have developed a model of IPS using a well-characterized murine BMT system (B10.BR-->CBA) in which lung injury after BMT can be induced by minor histocompatibility (H) antigenic differences between donor and host. Lung pathology and broncho-alveolar lavage (BAL) fluid were analyzed in transplant recipients before and after both syngeneic and allogeneic BMT. At 2 weeks after BMT, no specific pathologic abnormalities were noted; at 6 weeks, both pneumonitis and mononuclear cell infiltration around vessels and bronchioles were observed only in mice receiving allogeneic BMT. This injury was associated with elevated BAL fluid levels of endotoxin (lipopolysaccharide [LPS]), neutrophils, and tumor necrosis factor alpha. No pathologic organisms were isolated from the respiratory tract of any animal. We also tested the role of endotoxin in the development of this injury. Injection of LPS 6 weeks after transplantation caused profound lung injury only in mice with moderate graft-versus-host disease; dramatic increases in BAL neutrophils and tumor necrosis factor alpha were observed, with alveolar hemorrhage occurring in 4 of 12 of these mice but in no other group. We conclude that (1) this murine BMT system is a potentially useful model of clinical IPS; (2) minor H differences between donor and recipient can be important stimuli in the pathogenesis of IPS; and (3) endotoxin in BAL fluid is associated with lung injury, and excess endotoxin can cause the development of alveolar hemorrhage in this model.
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