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Detection of nitrative stress is crucial to understanding redox signaling and pathophysiology. Dysregulated nitrative stress, which generates high levels of peroxynitrite, can damage lipid membranes and cause activation of proinflammatory pathways associated with pulmonary complications. Here, we present a protocol for implementing a peroxynitrite-sensing phospholipid to investigate nitrative stress in murine cells and lung tissue. We detail procedures for sensing ONOO–in stimulated cells, both ex vivoand in vivo, using murine models of acute lung injury (ALI).

Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome, which is caused by mutations of the tumor suppressor gene folliculin(FLCN). The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcndeletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cremice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

Aquaporin 11 (AQP11) is a newly described member of the protein family of transport channels. AQP11 associates with the endoplasmic reticulum (ER) and is highly expressed in proximal tubular epithelial cells in the kidney. Previously, we identified and characterized a recessive mutation of the highly conserved Cys227 to Ser227 in mouse AQP11 that caused proximal tubule (PT) injury and kidney failure in mutant mice. The current study revealed induction of ER stress, unfolded protein response, and apoptosis as molecular mechanisms of this PT injury. Cys227Ser mutation interfered with maintenance of AQP11 oligomeric structure. AQP11 is abundantly expressed in the S1 PT segment, a site of major renal glucose flux, and Aqp11mutant mice developed PT-specific mitochondrial injury. Glucose increased AQP11 protein expression in wild-type kidney and upregulation of AQP11 expression by glucose in vitro was prevented by phlorizin, an inhibitor of sodium-dependent glucose transport across PT. Total AQP11 levels in heterozygotes were higher than in wild-type mice but were not further increased in response to glucose. In Aqp11insufficient PT cells, glucose potentiated increases in reactive oxygen species (ROS) production. ROS production was also elevated in Aqp11mutation carriers. Phenotypically normal mice heterozygous for the Aqp11mutation repeatedly treated with glucose showed increased blood urea nitrogen levels that were prevented by the antioxidant sulforaphane or by phlorizin. Our results indicate an important role for AQP11 to prevent glucose-induced oxidative stress in proximal tubules.

The lung is continuously exposed to inhaled pathogens (toxic pollutants, micro-organisms, environmental antigens, allergens) from the external environment. In the broncho-alveolar space, the critical balance between a measured protective response against harmful pathogens and an inappropriate inflammatory response to harmless particles is discerned by the innate pulmonary immune system. Among its many components, the surfactant proteins and specifically the pulmonary collectins (surfactant proteins A [SP-A] and D [SP-D]) appear to provide important contributions to the modulation of host defense and inflammation in the lung. Many studies have shown that multimerization of SP-A and SP-D are important for efficient local host defense including neutralization and opsonization of influenza A virus, binding Pneumocystis murina and inhibition of LPS-induced inflammatory cell responses. These observations strongly imply that oligomerization of collectins is a critical feature of its function. However, during the inflammatory state, despite normal pool sizes, chemical modification of collectins can result in alteration of their structure and function. Both pulmonary collectins can be altered through proteolytic inactivation, nitration, S-nitrosylation, oxidation and/or crosslinking as a consequence of the inflammatory milieu facilitated by cytokines, nitric oxide, proteases, and other chemical mediators released by inflammatory cells. Thus, this review will summarize recent developments in our understanding of the relationship between post-translational assembly of collectins and their modification by inflammation as an important molecular switch for the regulation of local innate host defense.

Alpha hemoglobin stabilizing protein (AHSP) reversibly binds nascent α globin to maintain its native structure and facilitate its incorporation into hemoglobin A. Previous studies indicate that some naturally occurring human α globinmutations may destabilize the protein by inhibiting its interactions with AHSP. However, these mutations could also affect hemoglobin A production through AHSP-independent effects, including reduced binding to β globin. We analyzed 6 human α globin variants with altered AHSP contact surfaces. Alpha globin amino acid substitutions H103Y, H103R, F117S, and P119S impaired interactions with both AHSP and β globin. These mutations are destabilizing in biochemical assays and are associated with microcytosis and anemia in humans. By contrast, K99E and K99N α globins bind β globin normally but exhibit attenuated binding to AHSP. These mutations impair protein folding and expression in vitro and appear to be mildly destabilizing in vivo. In Escherichia coliand erythroid cells, α globin K99E stability is rescued on coexpression with AHSP mutants in which binding to the abnormal globin chain is restored. Our results better define the biochemical properties of some α globin variants and support the hypothesis that AHSP promotes α globin chain stability during human erythropoiesis.

ABSTRACTSurfactant protein A (SP-A), a member of the collectin family, selectively binds to Pneumocystis cariniiand mediates interactions between pathogen and host alveolar macrophages in vitro. To test the hypothesis that mice lacking SP-A have delayed clearance of Pneumocystisorganisms and enhanced lung injury, wild-type C57BL/6 (WT) and SP-A-deficient mice (SP-A−/−) with or without selective CD4+-T-cell depletion were intratracheally inoculated with Pneumocystisorganisms. Four weeks later, CD4-depleted SP-A-deficient mice had developed a more severe Pneumocystisinfection than CD4-depleted WT (P. cariniipneumonia [PCP] scores of 3 versus 2, respectively). Whereas all non-CD4-depleted WT mice were free of PCP, intact SP-A−/−mice also had evidence of increased organism burden. Pneumocystisinfection in SP-A-deficient mice was associated histologically with enhanced peribronchial and/or perivascular cellularity (score of 4 versus 2, SP-A−/−versus C57BL/6 mice, respectively) and a corresponding increase in bronchoalveolar lavage (BAL) cell counts. Increases in SP-D content, gamma interferon, interleukin-4, interleukin-5, and tumor necrosis factor alpha in BAL fluid occurred but were attenuated in PCP-infected SP-A−/−mice compared to WT mice. There were increases in total BAL NO levels in both infected groups, but nitrite levels were higher in SP-A−/−mice, indicating a reduction in production of higher oxides of nitrogen that was also reflected in lower levels of 3-nitrotyrosine staining in the SP-A−/−group. We conclude that despite increases in inflammatory cells, SP-A-deficient mice infected with P. cariniiexhibit an enhanced susceptibility to the organism and attenuated production of proinflammatory cytokines and reactive oxygen-nitrogen species. These data support the concept that SP-A is a local effector molecule in the lung host defense against P. cariniiin vivo.

There is substantial evidence that proteinS-nitrosylation provides a significant route through which nitric oxide (NO)-derived bioactivity is conveyed. However, most examples of S-nitrosylation have been characterized on the basis of analysis in vitro, and relatively little progress has been made in assessing the participant forms of nitric-oxide synthase (NOS) or the dynamics of protein S-nitrosylationin situ. Here we utilize antibodies specific for the nitrosothiol (SNO) moiety to provide an immunohistochemical demonstration that protein S-nitrosylation is coupled to the activity of each of the major forms of NOS. In cultured endothelial cells, SNO-protein immunoreactivity increases in response to Ca2+-stimulated endothelial NOS (eNOS) activity, and in aortic rings, endothelium-derived and eNOS-mediated relaxation (EDRF) is coupled to increased protein S-nitrosylation in both endothelial and associated smooth muscle cells. In cultured macrophages, SNO-protein levels increase upon cytokine induction of induced NOS (iNOS), and in PC12 cells, increased proteinS-nitrosylation is linked to nerve growth factor induction of neuronal NOS (nNOS). In addition, we describe developmental and pathophysiological increases in SNO-protein immunoreactivity within human lung. These results, which demonstrate Ca2+, neurohumoral, growth factor, cytokine, and developmental regulation of protein S-nitrosylation that is coupled to NOS expression and activity, provide unique evidence for the proposition that this ubiquitous NO-derived post-translational protein modification serves as a major effector of NO-related bioactivity.

Nitric oxide (NO) has been shown to mediate a number of different physiological functions within every major organ system. This wide variety of functional roles is made all the more remarkable when one considers that NO is a simple diatomic molecule. However, despite the simplicity of the molecule, NO possesses a wide range of chemical reactivity and multiple potential reactive targets. It is the variability of NO reactivity, which leads to its capability to control such a vast range of biological functions. In essence the functionality of NO is controlled by its chemical reactivity. In order to understand this possibility further it is necessary to consider the biologically relevant reactions of nitric oxide. © 2001 Wiley-Liss, Inc.

Nitric oxide is a key regulator of diverse physiological processes that include vasoregulation, platelet and neutrophil adherence, and immune defense. The detection and quantification of nitric oxide has been accomplishd by a variety of methodologies. In this paper we desribe methodologies to detect nitric oxide in solution using an electrochemical detector and provide examples of nitric oxide detection in biological systems including data from plasma and cell suspensions. The experiments outlined show that electrochemical detection is suitable for detecting and quantifying nitric oxide within biological samples and buffers. Potential pitfalls and necessary controls are discussed. The use of electrochemical detection was also found to be useful in determining the flux of nitric oxide produced by donor species in different biological buffers.

The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitritemediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine‐containing proteins. Peroxynitrite‐modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration‐, time‐, and temperature‐dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.

The objective of this study was to investigate the mechanism of S-nitrosothiol formation under physiological conditions. A mechanism is proposed by which nitric oxide (·NO) reacts directly with reduced thiol to produce a radical intermediate, R-S-N·-O-H. This intermediate reduces an electron acceptor to produce S-nitrosothiol. Under aerobic conditions O2acts as the electron acceptor and is reduced to produce superoxide (o-.2. The following experimental evidence is provided in support of this mechanism. Cysteine accelerates the consumption of ·NO by 2.5-fold under physiological conditions. The consumption of O2in the presence of ·NO and cysteine is increased by 2.4-fold. The reaction orders of ·NO and cysteine are second and first order, respectively. The second order of reaction for ·NO may result from interaction between ·NO and Oo-.2to form peroxynitrite. In the presence of Cu,Zn-superoxide dismutase, the reaction of ·NO with cysteine generates hydrogen peroxide, indicating that the reaction generateso-.2. Finally, the formation of S-nitrosothiol is demonstrated in an anaerobic environment and, as predicted by the mechanism, is dependent on the presence of an electron acceptor. These results demonstrate that under physiological conditions ·NO reacts directly with thiols to form S-nitrosothiol in the presence of an electron acceptor.

Fatty acid nitroalkenes are reversibly-reactive electrophiles that are endogenously detectable at nM concentrations and display anti-inflammatory, pro-survival actions. These actions are elicited through the alteration of signal transduction proteins via a Michael addition on nucleophilic cysteine thiols. Nitrated fatty acids (NO2-FAs), like 9- or 10-nitro-octadec-9-enolic acid, will act on signal transduction proteins directly or on key regulatory proteins to cause an up-regulation or down-regulation of the protein’s expression, yielding an anti-inflammatory response. These responses have been characterized in many organ systems, such as the cardiovascular system, with the pulmonary system less well defined. Macrophages are one of the most abundant immune cells in the lung and are essential in maintaining lung homeostasis. Despite this, macrophages can play a role in both acute and chronic lung injury due to up-regulation of anti-inflammatory signal transduction pathways and down-regulation of pro-inflammatory pathways. Through their propensity to alter signal transduction pathways, NO2-FAs may be able to reduce macrophage activation during pulmonary injury. This review will focus on the implications of NO2-FAs on macrophage activation in the lung and the signal transduction pathways that may be altered, leading to reduced pulmonary injury.

In response to the NSW Tobacco Action Plan 2005?2009, Greater Southern Area Health Service (GSAHS) has developed a local plan. This short report describes how activities promoted in the state plan were prioritised and six outcomes identified as the focus for the GSAHS Tobacco Control Plan 2006?2009.

As the majority of the determinants of health are controlled outside the health system, the challenge for promoting health is to find a way of influencing these determinants. Health impact assessment was used in Bungendore, NSW, in an attempt to influence decisions relating to scenarios for urban development. Twelve months after the project was completed, interim evaluation has revealed evidence that the health impact assessment has had a positive effect on preliminary land-use planning work.

AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp-/- mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp-/- mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp-/- //alpha globin*a/aa) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.

Alpha hemoglobin stabilizing protein (AHSP) binds α hemoglobin (Hb) to stabilize its structure and limit its chemical reactivity in vitroand in vivo. Addition of β Hb to α Hb-AHSP displaces AHSP to form HbA (α2β2) tetramer. Recently, we determined that binding of AHSP to free α Hb generates a novel structure in which the helix F of α Hb is disordered and the heme iron is reduced to the ferrous form and coordinated by the distal histidine (ferrous α Hb-AHSP). Over time, the heme iron within this complex oxidizes to the ferric form (ferric α Hb-AHSP) and the F helix reorders, generating a bis-histidyl heme iron that exhibits decreased capacity to catalyze the formation of reactive oxygen species. At room temperature in ambient oxygen, the conversion of ferrous to ferric α Hb-AHSP occurs over several hours. We proposed that in vivo, these complexes could serve as transient intermediates to maintain α Hb in a stable state until β Hb is available to form tetrameric HbA. The current study addresses two questions related to this hypothesis:

Recent studies have suggested that binding of oxygen to hemoglobin (Hb) facilitates reactions of nitric oxide (NO) that lead to production of S-nitrosohemoglobin (SNO-Hb), and that vasodilator S-nitrosthiol (SNO) is dispensed by red blood cells (RBCs) at low oxygen tension (pO2) to dilate blood vessels. In human lungs, NO bioactivity serves to attenuate hypoxic pulmonary vasoconstriction (HPV). We therefore considered the possibility that RBC-SNO may oppose HPV and that defective vasodilation by RBCs may contribute to the pathophysiology of pulmonary arterial hypertension (PAH). Here we report that RBCs from patients with PAH exhibit substantial depletion of SNO-Hb and consequent impairment in hypoxia-mediated vasodilation. Furthermore, levels of RBC-NO correlated inversely with pulmonary artery pressures. A SNO-Hb deficiency characteristic of PAH was reproduced in control RBCs by hypoxia: loss of SNO-Hb was accompanied by a buildup of heme-NO species that are deficient in the pO2-governed intramolecular transfer of NO to cysteine thiol, yielding RBCs deficient in NO bioactivity. SNO-deficient RBCs produced exaggerated HPV responses as compared to SNO-replete RBCs. In PAH patients, SNO-Hb repletion fully restored the hypoxic vasodilator activity of RBCs. Our results suggest that a deficiency in RBC-SNO contributes to pulmonary hypertension and hypoxemia, and that repletion of RBC-SNO represents a rational strategy for treating PAH patients.