Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

A minimally invasive bronchoscopic approach for direct delivery to murine airways and application to models of pulmonary infection.

The laboratory mouse is used extensively for human disease modeling and preclinical therapeutic testing for efficacy, biodistribution, and toxicity. The variety of murine models available, and the ability to create new ones, eclipses all other species, but the size of mice and their organs create challenges for many in vivo studies. For pulmonary research, improved methods to access murine airways and lungs, and track substances administered to them, would be desirable. A nonsurgical endoscopic system with a camera, effectively a bronchoscope, coupled with a cryoimaging fluorescence microscopy technique to view the lungs in 3D, is described here that allows visualization of the procedure, including the anatomical location at which substances are instilled and fluorescence detection of those substances. We have applied it to bacterial infection studies to characterize better and optimize a chronic lung infection murine model in which we instill bacteria-laden agarose beads into the airways and lungs to extend the duration of the infection and inflammation. The use of the endoscope as guidance for placing a catheter into the airways is simple and quick, requiring only momentary sedation, and reduces post-procedural mortality compared with our previous instillation method that includes a trans-tracheal surgery. The endoscopic method improves speed and precision of delivery while reducing the stress on animals and the number of animals generated and used for experiments.

NOX4 Mediates Epithelial Cell Death in Hyperoxic Acute Lung Injury Through Mitochondrial Reactive Oxygen Species.

Management of acute respiratory distress involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). NADPH oxidase (NOX) could be a major source of reactive oxygen species (ROS) in hyperoxia (HO). Epithelial cell death is a crucial step in the development of many lung diseases. Alveolar type II (AT2) cells are the metabolically active epithelial cells of alveoli that serve as a source of AT1 cells following lung injury. The aim of this study was to determine the possible role of AT2 epithelial cell NOX4 in epithelial cell death from HALI. Wild type (WT), Nox4 fl/fl (control), and Nox4 -/- Spc-Cre mice were exposed to room air (NO) or 95% O2 (HO) to investigate the structural and functional changes in the lung. C57BL/6J WT animals subjected to HO showed increased expression of lung NOX4 compared to NO. Significant HALI, increased bronchoalveolar lavage cell counts, increased protein levels, elevated proinflammatory cytokines and increased AT2 cell death seen in hyperoxic Nox4 fl/fl control mice were attenuated in HO-exposed Nox4 -/- Spc-Cre mice. HO-induced expression of NOX4 in MLE cells resulted in increased mitochondrial (mt) superoxide production and cell apoptosis, which was reduced in NOX4 siRNA silenced cells. This study demonstrates a novel role for epithelial cell NOX4 in accelerating lung epithelial cell apoptosis from HALI. Deletion of the Nox4 gene in AT2 cells or silencing NOX4 in lung epithelial cells protected the lungs from severe HALI with reduced apoptosis and decreased mt ROS production in HO. These results suggest NOX4 as a potential target for the treatment of HALI.

Airway Thiol-NO Adducts as Determinants of Exhaled NO.

Thiol-NO adducts such as S-nitrosoglutathione (GSNO) are endogenous bronchodilators in human airways. Decreased airway S-nitrosothiol concentrations are associated with asthma. Nitric oxide (NO), a breakdown product of GSNO, is measured in exhaled breath as a biomarker in asthma; an elevated fraction of expired NO (FENO) is associated with asthmatic airway inflammation. We hypothesized that FENO could reflect airway S-nitrosothiol concentrations. To test this hypothesis, we first studied the relationship between mixed expired NO and airway S-nitrosothiols in patients endotracheally intubated for respiratory failure. The inverse (Lineweaver-Burke type) relationship suggested that expired NO could reflect the rate of pulmonary S-nitrosothiol breakdown. We thus studied NO evolution from the lungs of mice (GSNO reductase -/-) unable reductively to catabolize GSNO. More NO was produced from GSNO in the -/- compared to wild type lungs. Finally, we formally tested the hypothesis that airway GSNO increases FENO using an inhalational challenge model in normal human subjects. FENO increased in all subjects tested, with a median t1/2 of 32.0 min. Taken together, these data demonstrate that FENO reports, at least in part, GSNO breakdown in the lungs. Unlike GSNO, NO is not present in the lungs in physiologically relevant concentrations. However, FENO following a GSNO challenge could be a non-invasive test for airway GSNO catabolism.

Hyperoxia-induced S1P1 signaling reduced angiogenesis by suppression of TIE-2 leading to experimental bronchopulmonary dysplasia.

INTRODUCTION: We have earlier shown that hyperoxia (HO)-induced sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling contribute to bronchopulmonary dysplasia (BPD). S1P acts through G protein-coupled receptors, S1P1 through S1P5. Further, we noted that heterozygous deletion of S1pr1 ameliorated the HO-induced BPD in the murine model. The mechanism by which S1P1 signaling contributes to HO-induced BPD was explored. METHODS: S1pr1+/+ and S1pr1+/- mice pups were exposed to either room air (RA) or HO (75% oxygen) for 7 days from PN 1-7. Lung injury and alveolar simplification was evaluated. Lung protein expression was determined by Western blotting and immunohistochemistry (IHC). In vitro experiments were performed using human lung microvascular endothelial cells (HLMVECs) with S1P1 inhibitor, NIBR0213 to interrogate the S1P1 signaling pathway. RESULTS: HO increased the expression of S1pr1 gene as well as S1P1 protein in both neonatal lungs and HLMVECs. The S1pr1+/- neonatal mice showed significant protection against HO-induced BPD which was accompanied by reduced inflammation markers in the bronchoalveolar lavage fluid. HO-induced reduction in ANG-1, TIE-2, and VEGF was rescued in S1pr1+/- mouse, accompanied by an improvement in the number of arterioles in the lung. HLMVECs exposed to HO increased the expression of KLF-2 accompanied by reduced expression of TIE-2, which was reversed with S1P1 inhibition. CONCLUSION: HO induces S1P1 followed by reduced expression of angiogenic factors. Reduction of S1P1 signaling restores ANG-1/ TIE-2 signaling leading to improved angiogenesis and alveolarization thus protecting against HO-induced neonatal lung injury.

Bronchopulmonary Dysplasia and Pulmonary Hypertension. The Role of Smooth Muscle adh5.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification, airway hyperreactivity, and pulmonary hypertension. In our BPD model, we have investigated the metabolism of the bronchodilator and pulmonary vasodilator GSNO (S-nitrosoglutathione). We have shown the GSNO catabolic enzyme encoded by adh5 (alcohol dehydrogenase-5), GSNO reductase, is epigenetically upregulated in hyperoxia. Here, we investigated the distribution of GSNO reductase expression in human BPD and created an animal model that recapitulates the human data. Blinded comparisons of GSNO reductase protein expression were performed in human lung tissues from infants and children with and without BPD. BPD phenotypes were evaluated in global (adh5-/-) and conditional smooth muscle (smooth muscle/adh5-/-) adh5 knockout mice. GSNO reductase was prominently expressed in the airways and vessels of human BPD subjects. Compared with controls, expression was greater in BPD smooth muscle, particularly in vascular smooth muscle (2.4-fold; P = 0.003). The BPD mouse model of neonatal hyperoxia caused significant alveolar simplification, airway hyperreactivity, and right ventricular and vessel hypertrophy. Global adh5-/- mice were protected from all three aspects of BPD, whereas smooth muscle/adh5-/- mice were only protected from pulmonary hypertensive changes. These data suggest adh5 is required for the development of BPD. Expression in the pulmonary vasculature is relevant to the pathophysiology of BPD-associated pulmonary hypertension. GSNO-mimetic agents or GSNO reductase inhibitors, both of which are currently in clinical trials for other conditions, could be considered for further study in BPD.

CPAP protects against hyperoxia-induced increase in airway reactivity in neonatal mice.

BACKGROUND: Oxygen and continuous positive airway pressure (CPAP) are primary modes of respiratory support for preterm infants. Animal models, however, have demonstrated adverse unintended effects of hyperoxia and CPAP on lung development. We investigate the effects of combined neonatal hyperoxia and CPAP exposure on airway function and morphology in mice. METHODS: Newborn mice were exposed to hyperoxia (40% O2) 24 h/day for 7 consecutive days with or without daily (3 h/day) concomitant CPAP. Two weeks after CPAP and/or hyperoxia treatment ended, lungs were assessed for airway (AW) hyperreactivity and morphology. RESULTS: CPAP and hyperoxia exposure alone increased airway reactivity compared to untreated control mice. CPAP-induced airway hyperreactivity was associated with epithelial and smooth muscle proliferation. In contrast, combined CPAP and hyperoxia treatment no longer resulted in increased airway reactivity, which was associated with normalization of smooth muscle and epithelial proliferation to values similar to untreated mice. CONCLUSIONS: Our data suggest that the combination of CPAP and hyperoxia decreases the adverse consequences on airway remodeling of either intervention alone. The complex interaction between mechanical stretch (via CPAP) and hyperoxia exposure on development of immature airways has implications for the pathophysiology of airway disease in former preterm infants receiving non-invasive respiratory support. IMPACT: CPAP and mild hyperoxia exposure alone increase airway reactivity in the neonatal mouse model. In contrast, combined CPAP and hyperoxia no longer induce airway hyperreactivity. Combined CPAP and hyperoxia normalize smooth muscle and epithelial proliferation to control values. Interaction between CPAP-induced stretch and mild hyperoxia exposure on immature airways has important implications for airway pathophysiology in former preterm infants.

S-Nitrosylation of CHIP Enhances F508Del-CFTR Maturation.

S-nitrosothiols (SNOs) are endogenous signaling molecules that have numerous beneficial effects on the airway via cyclic guanosine monophosphate-dependent and -independent processes. Healthy human airways contain SNOs, but SNO levels are lower in the airways of patients with cystic fibrosis (CF). In this study, we examined the interaction between SNOs and the molecular cochaperone C-terminus Hsc70 interacting protein (CHIP), which is an E3 ubiquitin ligase that targets improperly folded CF transmembrane conductance regulator (CFTR) for subsequent degradation. Both CFBE41o- cells expressing either wild-type or F508del-CFTR and primary human bronchial epithelial cells express CHIP. Confocal microscopy and IP studies showed the cellular colocalization of CFTR and CHIP, and showed that S-nitrosoglutathione inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the mature and immature forms of F508del-CFTR. In fact, SNO inhibition of the function and expression of CHIP not only improved the maturation of CFTR but also increased CFTR's stability at the cell membrane. S-nitrosoglutathione-treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than cells that were not treated, suggesting that the S-nitrosylation of CHIP prevents the ubiquitination of CFTR by inhibiting CHIP's E3 ubiquitin ligase function. Furthermore, the exogenous SNOs S-nitrosoglutathione diethyl ester and S-nitro-N-acetylcysteine increased the expression of CFTR at the cell surface. After CHIP knockdown with siRNA duplexes specific for CHIP, F508del-CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del-CFTR expression on airway epithelial cell surfaces. Together, these findings indicate that S-nitrosylation of CHIP is a novel mechanism of CFTR correction, and we anticipate that these insights will allow different SNOs to be optimized as agents for CF therapy.

Linaclotide improves gastrointestinal transit in cystic fibrosis mice by inhibiting sodium/hydrogen exchanger 3.

Gastrointestinal dysfunction in cystic fibrosis (CF) is a prominent source of pain among patients with CF. Linaclotide, a guanylate cyclase C (GCC) receptor agonist, is a US Food and Drug Administration-approved drug prescribed for chronic constipation but has not been widely used in CF, as the cystic fibrosis transmembrane conductance regulator (CFTR) is the main mechanism of action. However, anecdotal clinical evidence suggests that linaclotide may be effective for treating some gastrointestinal symptoms in CF. The goal of this study was to determine the effectiveness and mechanism of linaclotide in treating CF gastrointestinal disorders using CF mouse models. Intestinal transit, chloride secretion, and intestinal lumen fluidity were assessed in wild-type and CF mouse models in response to linaclotide. CFTR and sodium/hydrogen exchanger 3 (NHE3) response to linaclotide was also evaluated. Linaclotide treatment improved intestinal transit in mice carrying either F508del or null Cftr mutations but did not induce detectable Cl- secretion. Linaclotide increased fluid retention and fluidity of CF intestinal contents, suggesting inhibition of fluid absorption. Targeted inhibition of sodium absorption by the NHE3 inhibitor tenapanor produced improvements in gastrointestinal transit similar to those produced by linaclotide treatment, suggesting that inhibition of fluid absorption by linaclotide contributes to improved gastrointestinal transit in CF. Our results demonstrate that linaclotide improves gastrointestinal transit in CF mouse models by increasing luminal fluidity through inhibiting NHE3-mediated sodium absorption. Further studies are necessary to assess whether linaclotide could improve CF intestinal pathologies in patients. GCC signaling and NHE3 inhibition may be therapeutic targets for CF intestinal manifestations. NEW & NOTEWORTHY Linaclotide's primary mechanism of action in alleviating chronic constipation is through cystic fibrosis transmembrane conductance regulator (CFTR), negating its use in patients with cystic fibrosis (CF). For the first time, our findings suggest that in the absence of CFTR, linaclotide can improve fluidity of the intestinal lumen through the inhibition of sodium/hydrogen exchanger 3. These findings suggest that linaclotide could improve CF intestinal pathologies in patients.

Long-term effects of recurrent intermittent hypoxia and hyperoxia on respiratory system mechanics in neonatal mice.

BACKGROUND: Premature infants are at increased risk for wheezing disorders. Clinically, these neonates experience recurrent episodes of apnea and desaturation often treated by increasing the fraction of inspired oxygen (FIO2). We developed a novel paradigm of neonatal intermittent hypoxia with subsequent hyperoxia overshoots (CIHO/E) and hypothesized that CIHO/E elicits long-term changes on pulmonary mechanics in mice. METHODS: Neonatal C57BL/6 mice received CIHO/E, which consisted of 10% O2 (1 min) followed by a transient exposure to 50% FIO2, on 10-min repeating cycles 24 h/d from birth to P7. Baseline respiratory mechanics, methacholine challenge, RT-PCR for pro and antioxidants, radial alveolar counts, and airway smooth muscle actin were assessed at P21 after 2-wk room air recovery. Control groups were mice exposed to normoxia, chronic intermittent hyperoxia (CIHE), and chronic intermittent hypoxia (CIHO). RESULTS: CIHO/E and CIHE increased airway resistance at higher doses of methacholine and decreased baseline compliance compared with normoxia mice. Lung mRNA for NOX2 was increased by CIHO/E. Radial alveolar counts and airway smooth muscle actin was not different between groups. CONCLUSION: Neonatal intermittent hypoxia/hyperoxia exposure results in long-term changes in respiratory mechanics. We speculate that recurrent desaturation with hyperoxia overshoot may increase oxidative stress and contribute to wheezing in former preterm infants.

The Effect of Continuous Positive Airway Pressure in a Mouse Model of Hyperoxic Neonatal Lung Injury.

BACKGROUND: Continuous positive airway pressure (CPAP) and supplemental oxygen have become the mainstay of neonatal respiratory support in preterm infants. Although oxygen therapy is associated with respiratory morbidities including bronchopulmonary dysplasia (BPD), the long-term effects of CPAP on lung function are largely unknown. We used a hyperoxia-induced mouse model of BPD to explore the effects of daily CPAP in the first week of life on later respiratory system mechanics. OBJECTIVE: We wanted to test the hypothesis that daily CPAP in a newborn-mouse model of BPD improves longer-term respiratory mechanics. METHODS: Mouse pups from C57BL/6 pregnant dams were exposed to room air (RA) or hyperoxia (50% O2, 24 h/day) for the first postnatal week with or without exposure to daily CPAP (6 cm H2O, 3 h/day). Respiratory system resistance (Rrs) and compliance (Crs) were measured following a subsequent 2-week period of RA recovery. Additional measurements included radial alveolar and macrophage counts. RESULTS: Mice exposed to hyperoxia had significantly elevated Rrs, decreased Crs, reduced alveolarization and increased macrophage counts at 3 weeks when compared to RA-treated mice. Daily CPAP treatment significantly improved Rrs, Crs and alveolarization and decreased lung macrophage infiltration in the hyperoxia-exposed pups. CONCLUSIONS: We have demonstrated that daily CPAP had a longer-term benefit on baseline respiratory system mechanics in a neonatal mouse model of BPD. We speculate that this beneficial effect of CPAP was the consequence of a decrease in the inflammatory response and resultant alveolar injury associated with hyperoxic lung injury in newborns.

Airway Hyperreactivity Is Delayed after Mild Neonatal Hyperoxic Exposure.

BACKGROUND: Wheezing disorders are prominent in former preterm infants beyond the neonatal period. OBJECTIVES: We used a neonatal mouse model to investigate the time course of airway hyperreactivity in response to mild (40% oxygen) or severe (70% oxygen) neonatal hyperoxia. METHODS: After hyperoxic exposure during the first week of postnatal life, we measured changes in airway reactivity using the in vitro living lung slice preparation at the end of exposure [postnatal day 8 (P8)] and 2 weeks later (P21). This was accompanied by measures of smooth muscle actin, myosin light chain (MLC) and alveolar morphology. RESULTS: Neither mild nor severe hyperoxia exposure affected airway reactivity to methacholine at P8 compared to normoxic controls. In contrast, airway reactivity was enhanced at P21 in mice exposed to mild (but not severe) hyperoxia, 2 weeks after exposure ended. This was associated with increased airway α-smooth muscle actin expression at P21 after 40% oxygen exposure without a significant increase in MLC. Alveolar morphology via radial alveolar counts was comparably diminished by both 40 and 70% oxygen at both P8 and P21. CONCLUSIONS: These data demonstrate that early mild hyperoxia exposure causes a delayed augmentation of airway reactivity, suggesting a long-term alteration in the trajectory of airway smooth muscle development and consistent with resultant symptomatology.

Anti-inflammatory effect of caffeine is associated with improved lung function after lipopolysaccharide-induced amnionitis.

BACKGROUND: Although caffeine enhances respiratory control and decreases the need for mechanical ventilation and resultant bronchopulmonary dysplasia, it may also have anti-inflammatory properties in protecting lung function. OBJECTIVE: We hypothesized that caffeine improves respiratory function via an anti-inflammatory effect in lungs of a lipopolysaccharide (LPS)-induced pro-inflammatory amnionitis rat pup model. METHODS: Caffeine was given orally (10 mg/kg/day) from postnatal day (p)1 to p14 to pups exposed to intra-amniotic LPS or normal saline. Expression of IL-1ß was assessed in lung homogenates at p8 and p14, and respiratory system resistance (Rrs) and compliance (Crs) as well as CD68 cell counts and radial alveolar counts were assessed at p8. RESULTS: In LPS-exposed rats, IL-1ß and CD68 cell counts both increased at p8 compared to normal saline controls. These increases in pro-inflammatory markers were no longer present in caffeine-treated LPS-exposed pups. Rrs was higher in LPS-exposed pups (4.7 ± 0.9 cm H2O/ml·s) at p8 versus controls (1.6 ± 0.3 cm H2O/ml·s, p < 0.01). LPS-exposed pups no longer exhibited a significant increase in Rrs (2.8 ± 0.5 cm H2O/ml·s) after caffeine. Crs did not differ significantly between groups, although radial alveolar counts were lower in both groups of LPS-exposed pups. CONCLUSIONS: Caffeine promotes anti-inflammatory effects in the immature lung of prenatal LPS-exposed rat pups associated with improvement of Rrs, suggesting a protective effect of caffeine on respiratory function via an anti-inflammatory mechanism.

Severity of neonatal hyperoxia determines structural and functional changes in developing mouse airway.

Wheezing is a major long-term respiratory morbidity in preterm infants with and without bronchopulmonary dysplasia. We hypothesized that mild vs. severe hyperoxic exposure in neonatal mice differentially affects airway smooth muscle hypertrophy and resultant airway reactivity. Newborn mice were exposed to 7 days of mild (40% oxygen) or severe (70% oxygen) hyperoxia vs. room air controls. Respiratory system resistance (Rrs), compliance (Crs), and airway reactivity were measured 14 days after oxygen exposure ended under ketamine/xylazine anesthesia. Baseline Rrs increased and Crs decreased in both treatment groups. Methacholine challenge dose dependently increased Rrs and decreased Crs in 40% oxygen-exposed mice, whereas Rrs and Crs responses were similar between 70% oxygen-exposed and normoxic controls. Airway smooth muscle thickness was increased in 40%- but not 70%-exposed mice, whereas collagen increased and both alveolar number and radial alveolar counts decreased after 40% and 70% oxygen. These data indicate that severity of hyperoxia may differentially affect structural and functional changes in the developing mouse airway that contribute to longer-term hyperreactivity. These findings may be important to our understanding of the complex role of neonatal supplemental oxygen therapy in postnatal development of airway responsiveness.

Lung inflammation induces IL-1ß expression in hypoglossal neurons in rat brainstem.

Perinatal inflammation is associated with respiratory morbidity. Immune modulation of brainstem respiratory control centers may provide a link for this pathobiology. We exposed 11-day old rats to intratracheal lipopolysaccharide (LPS, 0.5 µg/g) to test the hypothesis that intrapulmonary inflammation increases expression of the proinflammatory cytokine IL-1ß within respiratory-related brainstem regions. Intratracheal LPS resulted in a 32% increase in IL-1ß protein expression in the medulla oblongata. In situ hybridization showed increased intensity of IL-1ß mRNA but no change in neuronal numbers. Co-localization experiments showed that hypoglossal neurons express IL-1ß mRNA and immunostaining showed a 43% increase in IL-1ß protein-expressing cells after LPS exposure. LPS treatment also significantly increased microglial cell numbers though they did not express IL-1ß mRNA. LPS-induced brainstem expression of neuronal IL-1ß mRNA and protein may have implications for our understanding of the vulnerability of neonatal respiratory control in response to a peripheral proinflammatory stimulus.

Role of arginase in impairing relaxation of lung parenchyma of hyperoxia-exposed neonatal rats.

BACKGROUND: Prolonged exposure of immature lungs to hyperoxia contributes to neonatal lung injury and airway hyperreactivity. We have previously demonstrated that neonatal exposure of rat pups to ≥95% O2 impairs airway relaxation due to disruption of nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling. OBJECTIVE: We now hypothesize that these impaired relaxation responses are secondary to hyperoxia-induced upregulation of arginase, which competes with NO synthase for L-arginine. METHODS: Rat pups were exposed to moderate neonatal hyperoxia (50% O2) or room air for 7 days from birth. In additional hyperoxic and room air groups, exogenous L-arginine (300 mg/kg/day i.p.) or arginase inhibitor (Nω-hydroxy-nor-arginine, 30 mg/kg/day i.p.) were administered daily. After 7 days, animals were anesthetized and sacrificed either for preparation of lung parenchymal strips or lung perfusion. RESULTS: In response to electrical field stimulation (EFS), bethanechol-preconstricted lung parenchymal strips from hyperoxic pups exhibited significantly reduced relaxation compared to room air controls. Supplementation of L-arginine or arginase blockade restored hyperoxia-induced impairment of relaxation. Expression of arginase I in airway epithelium was increased in response to hyperoxia but reduced by arginase blockade. Arginase activity was also significantly increased in hyperoxic lungs as compared to room air controls and reduced following arginase blockade. EFS-induced production of NO was decreased in hyperoxia-exposed airway smooth muscle and restored by arginase blockade. CONCLUSION: These data suggest that NO-cGMP signaling is disrupted in neonatal rat pups exposed to even moderate hyperoxia due to increased arginase activity and consequent decreased bioavailability of the substrate L-arginine. We speculate that supplementation of arginine and/or inhibition of arginase may be a useful therapeutic tool to prevent or treat neonatal lung injury.

Role of brain-derived neurotrophic factor in hyperoxia-induced enhancement of contractility and impairment of relaxation in lung parenchyma.

Prolonged hyperoxic exposure contributes to neonatal lung injury, and airway hyperreactivity is characterized by enhanced contraction and impaired relaxation of airway smooth muscle. Our previous data demonstrate that hyperoxia in rat pups upregulates expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, disrupts NO-cGMP signaling, and impairs cAMP production in airway smooth muscle. We hypothesized that BDNF-tyrosine kinase B (TrkB) signaling plays a functional role in airway hyperreactivity via upregulation of cholinergic mechanisms in hyperoxia-exposed lungs. Five-day-old rat pups were exposed to >or=95% oxygen or room air for 7 days and administered daily tyrosine kinase inhibitor K-252a (50 microg x kg(-1) x day(-1) i.p.) to block BDNF-TrkB signaling or vehicle. Lungs were removed for HPLC measurement of ACh or for in vitro force measurement of lung parenchymal strips. ACh content doubled in hyperoxic compared with room air-exposed lungs. K-252a treatment of hyperoxic pups restored ACh content to room air levels. Hyperoxia increased contraction and impaired relaxation of lung strips in response to incremental electrical field stimulation. K-252a administration to hyperoxic pups reversed this increase in contraction and decrease in relaxation. K-252a or TrkB-Fc was used to block the effect of exogenous BDNF in vitro. Both K-252a and TrkB-Fc blocked the effects of exogenous BDNF. Hyperoxia decreased cAMP and cGMP levels in lung strips, and blockade of BDNF-TrkB signaling restored cAMP but not cGMP to control levels. Therefore, hyperoxia-induced increase in activity of BDNF-TrkB receptor signaling appears to play a critical role in enhancing cholinergically mediated contractile responses of lung parenchyma.

Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

Adenosine A2A receptors are expressed by GABAergic neurons of medulla oblongata in developing rat.

During early development, adenosine contributes to the occurrence of respiratory depression and recurrent apneas. Recent physiological studies indicate that GABAergic mechanisms may be involved in this inhibitory action of adenosine, via their A(2A) receptors. In the present study, in situ hybridization with ribonucleotide probes for A(2A) receptor (A(2A)R) mRNA was combined with the immunolabeling technique for parvalbumin and transneuronal retrograde tracing method using green fluorescent protein expressing pseudorabies virus (GFP-PRV) to (1) characterize age-dependent changes in the expression of adenosine A(2A)Rs mRNA in brain stem regions where GABAergic neurons are located; (2) determine whether GABA-containing neurons express A(2A)R mRNA traits, and (3) identify whether bulbospinal GABAergic neurons projecting to phrenic nuclei contain A(2A)R mRNA. Results revealed expression of A(2A) receptors in regions of medulla oblongata containing GABAergic neurons, namely in the ventral aspect of the medulla, within the Bötzinger region and caudal to it, the gigantocellular reticular nucleus, midline neurons and the caudal ventrolateral medulla oblongata. Furthermore, a subpopulation of identified GABAergic neurons, projecting to the phrenic motor nuclei, possess A(2A)R mRNA. It is concluded that adenosine A(2A)Rs expressed by GABAergic neurons are likely to play a role in mediating adenosine-induced respiratory depression.

Airway-related vagal preganglionic neurons express brain-derived neurotrophic factor and TrkB receptors: implications for neuronal plasticity.

Recent evidence indicates that brain-derived neurotrophic factor (BDNF) is present in neurons and may affect neurotransmitter release, cell excitability, and synaptic plasticity via activation of tyrosine kinase B (TrkB) receptors. However, whether airway-related vagal preganglionic neurons (AVPNs) produce BDNF and contain TrkB receptors is not known. Hence, in ferrets, we examined BDNF and TrkB receptor expression in identified AVPNs using in situ hybridization and immunohistochemistry. BDNF protein levels were measured within the rostral nucleus ambiguus (rNA) region by ELISA. We observed that the subpopulation of AVPNs, identified by neuroanatomical tract tracing, within the rNA region express BDNF mRNA, BDNF protein, as well as TrkB receptor. In addition, brain tissue from the rNA region contained measurable amounts of BDNF that were comparable to the hippocampal region of the brain. These data indicate, for the first time, that the BDNF-TrkB system is expressed by AVPNs and may play a significant role in regulating cholinergic outflow to the airways.