S-nitrosothiol repletion by an inhaled gas regulates pulmonary function

NO synthases are widely distributed in the lung and are extensively involved in the control of airway and vascular homeostasis. It is recognized, however, that the O2-rich environment of the lung may predispose NO toward toxicity. These Janus faces of NO are manifest in recent clinical trials with inhaled NO gas, which has shown therapeutic benefit in some patient populations but increased morbidity in others. In the airways and circulation of humans, most NO bioactivity is packaged in the form of S-nitrosothiols (SNOs), which are relatively resistant to toxic reactions with O2/O\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document}. This finding has led to the proposition that channeling of NO into SNOs may provide a natural defense against lung toxicity. The means to selectively manipulate the SNO pool, however, has not been previously possible. Here we report on a gas, O-nitrosoethanol (ENO), which does not react with O2 or release NO and which markedly increases the concentration of indigenous species of SNO within airway lining fluid. Inhalation of ENO provided immediate relief from hypoxic pulmonary vasoconstriction without affecting systemic hemodynamics. Further, in a porcine model of lung injury, there was no rebound in cardiopulmonary hemodynamics or fall in oxygenation on stopping the drug (as seen with NO gas), and additionally ENO protected against a decline in cardiac output. Our data suggest that SNOs within the lung serve in matching ventilation to perfusion, and can be manipulated for therapeutic gain. Thus, ENO may be of particular benefit to patients with pulmonary hypertension, hypoxemia, and/or right heart failure, and may offer a new therapeutic approach in disorders such as asthma and cystic fibrosis, where the airways may be depleted of SNOs.

Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Chronic Pseudomonas aeruginosa infection occurs in 75–90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108–117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108–117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant ΔF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Lack of tissue glucocorticoid reactivation in 11β-hydroxysteroid dehydrogenase type 1 knockout mice ameliorates age-related learning impairments

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) intracellularly regenerates active corticosterone from circulating inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The hippocampus is a brain structure particularly vulnerable to glucocorticoid neurotoxicity with aging. In intact hippocampal cells in culture, 11β-HSD-1 acts as a functional 11β-reductase reactivating inert 11-DHC to corticosterone, thereby potentiating kainate neurotoxicity. We examined the functional significance of 11β-HSD-1 in the central nervous system by using knockout mice. Aged wild-type mice developed elevated plasma corticosterone levels that correlated with learning deficits in the watermaze. In contrast, despite elevated plasma corticosterone levels throughout life, this glucocorticoid-associated learning deficit was ameliorated in aged 11β-HSD-1 knockout mice, implicating lower intraneuronal corticosterone levels through lack of 11-DHC reactivation. Indeed, aged knockout mice showed significantly lower hippocampal tissue corticosterone levels than wild-type controls. These findings demonstrate that tissue corticosterone levels do not merely reflect plasma levels and appear to play a more important role in hippocampal functions than circulating blood levels. The data emphasize the crucial importance of local enzymes in determining intracellular glucocorticoid activity. Selective 11β-HSD-1 inhibitors may protect against hippocampal function decline with age.

Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis

To determine the importance of mitochondrial reactive oxygen species toxicity in aging and senescence, we analyzed changes in mitochondrial function with age in mice with partial or complete deficiencies in the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Liver mitochondria from homozygous mutant mice, with a complete deficiency in MnSOD, exhibited substantial respiration inhibition and marked sensitization of the mitochondrial permeability transition pore. Mitochondria from heterozygous mice, with a partial deficiency in MnSOD, showed evidence of increased proton leak, inhibition of respiration, and early and rapid accumulation of mitochondrial oxidative damage. Furthermore, chronic oxidative stress in the heterozygous mice resulted in an increased sensitization of the mitochondrial permeability transition pore and the premature induction of apoptosis, which presumably eliminates the cells with damaged mitochondria. Mice with normal MnSOD levels show the same age-related mitochondrial decline as the heterozygotes but occurring later in life. The premature decline in mitochondrial function in the heterozygote was associated with the compensatory up-regulation of oxidative phosphorylation enzyme activity. Thus mitochondrial reactive oxygen species production, oxidative stress, functional decline, and the initiation of apoptosis appear to be central components of the aging process.

Use of intrinsic modes in biology: Examples of indicial response of pulmonary blood pressure to ± step hypoxia

Recently, a new method to analyze biological nonstationary stochastic variables has been presented. The method is especially suitable to analyze the variation of one biological variable with respect to changes of another variable. Here, it is illustrated by the change of the pulmonary blood pressure in response to a step change of oxygen concentration in the gas that an animal breathes. The pressure signal is resolved into the sum of a set of oscillatory intrinsic mode functions, which have zero “local mean,” and a final nonoscillatory mode. With this device, we obtain a set of “mean trends,” each of which represents a “mean” in a definitive sense, and together they represent the mean trend systematically with different degrees of oscillatory content. Correspondingly, the oscillatory content of the signal about any mean trend can be represented by a set of partial sums of intrinsic mode functions. When the concept of “indicial response function” is used to describe the change of one variable in response to a step change of another variable, we now have a set of indicial response functions of the mean trends and another set of indicial response functions to describe the energy or intensity of oscillations about each mean trend. Each of these can be represented by an analytic function whose coefficients can be determined by a least-squares curve-fitting procedure. In this way, experimental results are stated sharply by analytic functions.

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines

Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium (5 μM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or orresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms.

Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into β-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by β-strand exchange between the reactive loop of one molecule and β-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin α1-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into β-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma α1-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55–Pro mutation.

Nonlinear indicial response of complex nonstationary oscillations as pulmonary hypertension responding to step hypoxia

This paper is devoted to the quantization of the degree of nonlinearity of the relationship between two biological variables when one of the variables is a complex nonstationary oscillatory signal. An example of the situation is the indicial responses of pulmonary blood pressure (P) to step changes of oxygen tension (ΔpO2) in the breathing gas. For a step change of ΔpO2 beginning at time t1, the pulmonary blood pressure is a nonlinear function of time and ΔpO2, which can be written as P(t-t1 | ΔpO2). An effective method does not exist to examine the nonlinear function P(t-t1 | ΔpO2). A systematic approach is proposed here. The definitions of mean trends and oscillations about the means are the keys. With these keys a practical method of calculation is devised. We fit the mean trends of blood pressure with analytic functions of time, whose nonlinearity with respect to the oxygen level is clarified here. The associated oscillations about the mean can be transformed into Hilbert spectrum. An integration of the square of the Hilbert spectrum over frequency yields a measure of oscillatory energy, which is also a function of time, whose mean trends can be expressed by analytic functions. The degree of nonlinearity of the oscillatory energy with respect to the oxygen level also is clarified here. Theoretical extension of the experimental nonlinear indicial functions to arbitrary history of hypoxia is proposed. Application of the results to tissue remodeling and tissue engineering of blood vessels is discussed.

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Δdnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Δdnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.

Altered stability of pulmonary surfactant in SP-C-deficient mice

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (−/−) mice were produced. The SP-C (−/−) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (−/−) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (−/−) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (−/−) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (−/−) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (−/−) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor-α signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Leukemia inhibitory factor induces differentiation of pituitary corticotroph function: an immuno-neuroendocrine phenotypic switch.

Leukemia inhibitory factor (LIF) promotes differentiated cell function in several systems. We recently reported LIF and LIF receptor expression in human fetal pituitary corticotrophs in vivo and demonstrated LIF stimulation of adrenocorticotrophin (ACTH) transcription in vitro, suggesting a role for LIF in corticotroph development. We therefore assessed the action of LIF on proliferating murine corticotroph cells (AtT20). LIF impairs proliferation of AtT20 cells (25% reduction versus control, P < 0.03), while simultaneously enhancing ACTH secretion (2-fold, P < 0.001) and augmenting ACTH responsiveness to corticotrophin-releasing hormone (CRH) action (4-fold, P < 0.001). This attenuation of cell growth is due to a block of cell cycle progression from G1 into S phase, as measured by flow cytometric analysis (24 +/- 0.8 versus 11.57 +/- 1.5, P < 0.001). Using bromodeoxyuridine incorporation assays, loss of cells in S phase was confirmed (25 +/- 0.08 to 9.4 +/- 1.4, P < 0.008). In contrast, CRH induced the G2/M phase (3.6 +/- 0.2 to 15.4 +/- 3, P < 0.001). This effect was blunted by LIF (P < 0.001 versus CRH alone). Cyclin A mRNA levels, which decline in S phase, were stimulated 3.5-fold by LIF and markedly suppressed by CRH. These results indicate a LIF-induced cell cycle block occurring at G1/S in corticotroph cells. Thus, LIF reduces proliferation, enhances ACTH secretion, and potentiates effects of CRH on ACTH secretion while blocking effects of CRH on the cell cycle. Responses of these three markers of differentiated corticotroph function indicate LIF to be a differentiation factor for pituitary corticotroph cells by preferential phenotypic switching from proliferative to synthetic.

Two cystic fibrosis transmembrane conductance regulator mutations have different effects on both pulmonary phenotype and regulation of outwardly rectified chloride currents.

Cystic fibrosis (CF), a disorder of electrolyte transport manifest in the lungs, pancreas, sweat duct, and vas deferens, is caused by mutations in the CF transmembrane conductance regulator (CFTR). The CFTR protein has been shown to function as a cAMP-activated chloride channel and also regulates a separate protein, the outwardly rectifying chloride channel (ORCC). To determine the consequence of disease-producing mutations upon these functions, mutant CFTR was transiently expressed in Xenopus oocytes and in human airway epithelial cells lacking functional CFTR. Both G551D, a mutation that causes severe lung disease, and A455E, a mutation associated with mild lung disease, altered but did not abolish CFTR's function as a chloride channel in Xenopus oocytes. Airway epithelial cells transfected with CFTR bearing either A455E or G551D had levels of chloride conductance significantly greater than those of mock-transfected and lower than those of wild-type CFTR-transfected cells, as measured by chloride efflux. A combination of channel blockers and analysis of current-voltage relationships were used to dissect the contribution of CFTR and the ORCC to whole cell currents of transfected cells. While CFTR bearing either mutation could function as a chloride channel, only CFTR bearing A455E retained the function of regulating the ORCC. These results indicate that CF mutations can affect CFTR functions differently and suggest that severity of pulmonary disease may be more closely associated with the regulatory rather than chloride channel function of CFTR.