bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ras in metal-induced EGF receptor signaling and NF-κB activation in human airway epithelial cells
Authors: Wu Weidong , Jaspers Ilona , Zhang Wenli , Graves Lee M. , Samet James M.
Published: March 2015

We showed previously that epithelial growth factor (EGF) receptor (EGFR) signaling is triggered by metallic compounds associated with ambient air particles. Specifically, we demonstrated that As, Zn, and V activated the EGFR tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this study, we examined the role of Ras in EGFR signaling and the nuclear factor-κB (NF-κB) activation pathway and the possible interaction between these two signaling pathways in a human airway epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal significantly increased Ras activity, and this effect was inhibited by the EGFR tyrosine kinase activity inhibitor PD-153035. Adenoviral-mediated overexpression of a dominant-negative mutant form of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced IκBα serine phosphorylation, IκBα breakdown, and NF-κB DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced IκBα breakdown and NF-κB activation, while inhibition of MEK activity with PD-98059 failed to do so. In summary, exposure to As, Zn, and V initiated EGFR signaling and Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced Ras-dependent NF-κB nuclear translocation. EGFR signaling appears to cross talk with NF-κB signaling at the level of Ras, but additional signals appear necessary for NF-κB activation. Together, these data suggest that, in V-treated BEAS-2B cells, Ras-dependent signaling is essential, but not sufficient, for activation of NF-κB.
Feeble bronchomotor responses in diabetic rats in association with decreased sensory neuropeptide release
Authors: Szilvássy Judith , Sziklai István , Horvath Peter , Szilasi Maria , Németh József , Kovács Péter , Szilvássy Zoltán
Published: March 2015

Type I diabetes is associated with a low incidence of asthma. We tested whether a decrease in sensory neuropeptide release is associated with an attenuated bronchoconstrictive response to field stimulation (FS; 100 stimuli, 20 V, 0.1 ms, 20 Hz) in streptozotocin (STZ)-induced diabetes. The organ fluid of the preparations were also tested for substance P, calcitonin gene-related peptide (CGRP), and somatostatin concentrations by RIA. Preparations were from either normal rats or those pretreated with 50 mg/kg STZ iv 8 wk before experiment. A group of STZ-treated animals was supplied with insulin delivery (4 IU/day sc) implants between 4 and 8 wk. A subgroup was formed to study the effect of capsaicin desensitization. The atropine-resistant contraction was attenuated by diabetes without capsaicin-sensitive relaxation response. Exogenous CGRP and substance P potentiated, whereas somatostatin inhibited (1 nM-10 μM) the FS-induced contractions in rings from either group. FS released somatostatin, CGRP, and substance P from 0.17 ± 0.024, 0.15 ± 0.022, and 1.65 ± 0.093 to 0.58 ± 0.032, 0.74 ± 0.122, and 5.34 ± 0.295 in preparations from normal, and from 0.19 ± 0.016, 0.11 ± 0.019, and 0.98 ± 0.116 to 0.22 ± 0.076, 0.34 ± 0.099, and 1.84 ± 0.316 fmol/mg wet wt in preparations from diabetic rats. Insulin supplementation restored neuropeptide release in rings from STZ-treated rats. The results show that the decreased FS-induced contractions occurred with a decrease in sensory neuropeptide release in STZ-diabetic rats.
PAR-2 activation, PGE2, and COX-2 in human asthmatic and nonasthmatic airway smooth muscle cells
Authors: Chambers Linda S. , Black Judith L. , Ge Qi , Carlin Stephen M. , Au Wendy W. , Poniris Maree , Thompson Joanne , Johnson Peter R. , Burgess Janette K.
Published: March 2015

The protease-activated receptor-2 (PAR-2) is present on human airway smooth muscle (ASM) cells and can be activated by mast cell tryptase, trypsin, or an activating peptide (AP). Trypsin induced significant increases in PGE2 release from human ASM cells after 6 and 24 h and also induced cyclooxygenase (COX)-2 mRNA expression and COX-2 protein. Tryptase and the PAR-2 AP did not alter PGE2 release or COX-2 protein levels, suggesting a lack of PAR-2 involvement. When we compared results in asthmatic and nonasthmatic muscle cells, both trypsin and bradykinin induced less PGE2 from asthmatic ASM cells, and bradykinin induced significantly less COX-2 mRNA in asthmatic cells. Significantly less PGE2 was released from proliferating ASM cells from asthmatic patients. In conclusion, trypsin induces PGE2 release and COX-2 in human ASM cells, which is unlikely to be via PAR-2 activation. In addition, ASM cells from asthmatic patients produce significantly less PGE2 and COX-2 compared with nonasthmatic cells. These findings may contribute to the increase in muscle mass evident in asthmatic airways.
Membrane currents in canine bronchial artery and their regulation by excitatory agonists
Authors: Li Q. J. , Janssen L. J.
Published: March 2015

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K+current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10−6M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca2+-activated K+current. Consistent with this, the K+current was markedly increased by raising external Ca2+to 4 mM but was decreased by nifedipine (10−6M) or by removing external Ca2+. When K+currents were blocked (by Cs+in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca2+current as follows: 1) activation that was voltage dependent (threshold and maximal at −50 and −10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of −26 ± 22 mV); and 3) blockade by nifedipine (10−6M). The thromboxane mimetic U-46619 (10−6M) caused a marked augmentation of outward K+current (as did 10 mM caffeine) lasting only 10–20 s; this was followed by significant suppression of the K+current lasting several minutes. Phenylephrine (10−4M) also suppressed the K+current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca2+-dependent K+channels and voltage-dependent Ca2+channels and that its excitation does not involve activation of Cl−channels.
Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse
Authors: Balasubramaniam Vivek , Tang Jen-Ruey , Maxey Anne , Plopper Charles G. , Abman Steven H.
Published: March 2015

In addition to its vasodilator properties, nitric oxide (NO) promotes angiogenesis in the systemic circulation and tumors. However, the role of NO in promoting normal lung vascular growth and its impact on alveolarization during development or in response to perinatal stress is unknown. We hypothesized that NO modulates lung vascular and alveolar growth and that decreased NO production impairs distal lung growth in response to mild hypoxia. Litters of 1-day-old mouse pups from parents that were heterozygous for endothelial nitric oxide synthase (eNOS) deficiency were placed in a hypobaric chamber at a simulated altitude of 12,300 ft (FiO2= 0.16). After 10 days, the mice were killed, and lungs were fixed for morphometric and molecular analysis. Compared with wild-type controls, mean linear intercept (MLI), which is inversely proportional to alveolar surface area, was increased in the eNOS-deficient (eNOS −/−) mice [51 ± 2 μm (eNOS −/−) vs. 41 ± 1 μm (wild type); P < 0.01]. MLI was also increased in the eNOS heterozygote (+/−) mice (44 ± 1 μm; P < 0.03 vs. wild type). Vascular volume density was decreased in the eNOS −/− mice compared with wild-type controls ( P < 0.03). Lung vascular endothelial growth factor (VEGF) protein and VEGF receptor-1 (VEGFR-1) protein content were not different between the study groups. In contrast, lung VEGFR-2 protein content was decreased from control values by 63 and 34% in the eNOS −/− and eNOS +/− mice, respectively ( P < 0.03). We conclude that exposure to mild hypoxia during a critical period of lung development impairs alveolarization and reduces vessel density in the eNOS-deficient mouse. We speculate that NO preserves normal distal lung growth during hypoxic stress, perhaps through preservation of VEGFR-2 signaling.
Dexamethasone-induced changes in lung function are not prevented by concomitant treatment with retinoic acid
Authors: Srinivasan Ganesh , Bruce Eugene N. , Houtz Pamela K. , Bruce Margaret C.
Published: March 2015

Alveolarization is impaired in rats treated with dexamethasone (Dex) on postnatal days 4–13, but concomitant treatment with all- trans retinoic acid (RA) increases alveolar number. To determine whether morphological changes induced by Dex and/or RA predict changes in lung function at 1 mo, we assessed resting breathing parameters, dynamic compliance, ventilation required to maintain O2 saturation at ≥90%, and pressure-volume curves of air-filled lungs. During resting breathing, mean tidal volume per gram was greater in Dex + RA-treated rats than in controls ( P < 0.05). Dynamic compliance was also greater in Dex- and Dex + RA-treated rats than in controls or RA-treated rats ( P < 0.02). In Dex- and Dex + RA-treated rats, we observed increased hysteresis ratios ( P ≤ 0.006), air trapping ( P < 0.05), and lung volumes at 5 and 13.5 cmH2O pressure ( P < 0.001) and decreased elastic recoil ( P < 0.007). The effect of Dex on elastic recoil was greater in female than in male rats ( P = 0.006). Despite impaired septation, O2saturation was not compromised in Dex- or Dex + RA-treated rats. Thus lung function changes induced by Dex treatment during alveolarization were not prevented by concomitant treatment with RA.
Glucocorticoid effects on vitamin K-dependent carboxylase activity and matrix Gla protein expression in rat lung
Authors: Gilbert Kirk A. , Rannels Stephen R.
Published: March 2015

The role of glucocorticoids in the regulation of vitamin K-dependent carboxylase activity was investigated in fetal and adult lung. Glucocorticoid deficiency induced by adrenalectomy (ADX) stimulated adult lung growth and reduced carboxylation in a tissue-specific manner. Type II epithelial cells were enriched in carboxylase activity, where ADX-induced downregulation was retained in freshly isolated cells. Carboxylase activity in fetal type II cells was one-half that found in fetal fibroblasts isolated from the same lungs, and both populations increased activity with time in culture. Both carboxylase activity and formation of γ-carboxyglutamate (Gla)-containing proteins were stimulated by dexamethasone (Dex) in fetal type II cells. Matrix Gla protein (MGP), a vitamin K-dependent protein known to be synthesized in type II cells, was also found in fetal fibroblasts, where its expression was stimulated by Dex. These combined results suggested an important role for glucocorticoids and MGP in the developing lung, where both epithelial and mesenchymal cells coordinate precise control of branching morphogenesis. We investigated MGP expression and its regulation by Dex in the fetal lung explant model. MGP mRNA and protein were increased in parallel with the formation of highly branched lungs, and this increase was stimulated twofold by Dex at each day of culture. Dex-treated explants were characterized by large, dilated, conducting airways and a peripheral rim of highly branched saccules compared with uniformly branched controls. We propose that glucocorticoids are important regulators of vitamin K function in the developing and adult lung.
Dexamethasone suppresses iNOS yet induces GTPCH and CAT-2 mRNA expression in rat lungs
Authors: Skimming Jeffrey W. , Nasiroglu Omer , Huang Chun-Jen , Wood Charles E. , Stevens Bruce R. , Haque Ikram U. L. , Scumpia Philip O. , Sarcia Paul J.
Published: March 2015

The in vivo mechanisms by which glucocorticoids inhibit nitric oxide expression await detailed investigation. In cell culture experiments, glucocorticoids have been shown to inhibit inducible nitric oxide synthase (iNOS) formation and activity. Glucocorticoids can inhibit iNOS activity in cultured cells by blocking arginine transport and inhibiting tetrahydrobiopterin biosynthesis. We recently reported that changes in intrapulmonary formation of nitric oxide in endotoxemic rats correspond with changes in transcription of the predominant arginine transporter cationic amino acid transporter (CAT)-2. Realizing that hemorrhagic shock induces nitric oxide overproduction in intact animals, we sought to explore whether glucocorticoids attenuate hemorrhagic shock-induced increases in intrapulmonary nitric oxide formation and whether they might do so by inhibiting the formation of tetrahydrobiopterin, iNOS protein, and CAT-2. We randomly assigned 10 male Sprague-Dawley rats to receive dexamethasone or normal saline. Bleeding the animals to a mean systemic blood pressure of between 40 and 45 mmHg created the hemorrhagic shock. Dexamethasone abrogated the increase in exhaled nitric oxide concentrations caused by hemorrhagic shock. At the end of the experiment, plasma nitrate/nitrite values were lower in the dexamethasone group than in the control group. The iNOS protein concentrations were also lower in the dexamethasone group than in the control group. Dexamethasone decreased the intrapulmonary iNOS mRNA concentrations yet increased both guanosine triphosphate cyclohydrolase I mRNA and CAT-2 mRNA. Our results support the idea that dexamethasone inhibits nitric oxide formation in a manner that is independent of tetrahydrobiopterin and arginine transport yet dependent on downregulation of iNOS mRNA expression.
Pulmonary type II cell hypertrophy and pulmonary lipoproteinosis are features of chronic IL-13 exposure
Authors: Homer Robert J. , Zheng Tao , Chupp Geoff , He Susan , Zhu Zhou , Chen Quingshen , Ma Bing , Hite R. Duncan , Gobran Laurice I. , Rooney Seamus A. , Elias Jack A.
Published: March 2015

Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.
STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation
Authors: Xu Weiling , Comhair Suzy A. A. , Zheng Shuo , Chu Shan C. , Marks-Konczalik Joanna , Moss Joel , Haque S. Jaharul , Erzurum Serpil C.
Published: March 2015

Interferon-γ (IFN-γ) is required for induction of the human nitric oxide synthase-2 ( NOS2) gene in lung epithelium. Although the human NOS2 promoter region contains many cytokine-responsive elements, the molecular basis of induction is only partially understood. Here, the major cis-regulatory elements that control IFN-γ-inducible NOS2 gene transcription in human lung epithelial cells are identified as composite response elements that bind signal transducer and activator of transcription 1 (STAT-1) and activator protein 1 (AP-1), which is comprised of c-Fos, Fra-2, c-Jun, and JunD. Notably, IFN-γ activation of the human NOS2 promoter is shown to require functional AP-1 regulatory region(s), suggesting a role for AP-1 activation/binding in the IFN-γ induction of genes. We show that c-Fos interacts with STAT-1 after IFN-γ activation and the c-Fos/STAT-1 complex binds to the γ-activated site (GAS) element in close proximity to AP-1 sites located at 4.9 kb upstream of the transcription start site. Taken together, our findings support a model in which a physical interaction between c-Fos and STAT-1 participates in NOS2 gene transcriptional activation.
Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation
Authors: Kohri Kazuhiro , Ueki Iris F. , Nadel Jay A.
Published: March 2015

Neutrophil products are implicated in hypersecretory airway diseases. To determine the mechanisms linking a proteolytic effect of human neutrophil elastase (HNE) and mucin overproduction, we examined the effects of HNE on MUC5AC mucin production in human airway epithelial (NCI-H292) cells. Stimulation with HNE for 5–30 min induced MUC5AC production 24 h later, which was prevented by HNE serine active site inhibitors, implicating a proteolytic effect of HNE. MUC5AC induction was preceded by epidermal growth factor receptor (EGFR) tyrosine phosphorylation and was prevented by selective EGFR tyrosine kinase inhibitors, implicating EGFR activation. HNE-induced MUC5AC production was inhibited by a neutralizing transforming growth factor-α (TGF-α, an EGFR ligand) antibody and by a neutralizing EGFR antibody but not by oxygen free radical scavengers, further implicating TGF-α and ligand-dependent EGFR activation in the response. HNE decreased pro-TGF-α in NCI-H292 cells and increased TGF-α in cell culture supernatant. From these results, we conclude that HNE-induced MUC5AC mucin production occurs via its proteolytic activation of an EGFR signaling cascade involving TGF-α.
Role of interleukin-1 in the pulmonary immune response duringPseudomonas aeruginosapneumonia
Authors: Schultz Marc J. , Rijneveld Anita W. , Florquin Sandrine , Edwards Carl K. , Dinarello Charles A. , van der Poll Tom
Published: March 2015

Pneumonia is associated with elevated concentrations of the proinflammatory cytokine interleukin (IL)-1 in the pulmonary compartment. To study the role of IL-1 in the pathogenesis of Pseudomonas pneumonia, IL-1 receptor type 1 gene-deficient ( IL-1R −/−) mice and wild-type mice were intranasally inoculated with Pseudomonas aeruginosa. The absence of the IL-1 signal attenuated the outgrowth of Pseudomonas in lungs, as reflected by an increasing number of colony-forming units (cfu) during Pseudomonaspneumonia in wild-type mice and a concurrently decreasing number of cfu during pulmonary infection in IL-1R −/− mice ( P < 0.05, IL-1R −/− mice vs. wild-type mice). Influx of neutrophils was decreased in bronchoalveolar lavage fluids in IL-1R −/− mice compared with wild-type mice. Similarly, lung levels of cytokines (tumor necrosis factor-α, IL-6) and chemokines (macrophage inflammatory protein-2 and KC) were lower in IL-1R −/− mice 24 h postinoculation. Consistent with results obtained in IL-1R −/− mice, treatment of wild-type mice with IL-1R antagonist also diminished outgrowth of Pseudomonas when compared with wild-type mice treated with vehicle ( P < 0.05). These results demonstrate that an absence or reduction in endogenous IL-1 activity improves host defense against Pseudomonas pneumonia while suppressing the inflammatory response.
Rho protein inactivation induced apoptosis of cultured human endothelial cells
Authors: Hippenstiel Stefan , Schmeck Bernd , N'Guessan Phillipe Dje , Seybold Joachim , Krüll Matthias , Preissner Klaus , Eichel-Streiber Christoph V. , Suttorp Norbert
Published: March 2015

Small GTP-binding Rho GTPases regulate important signaling pathways in endothelial cells, but little is known about their role in endothelial cell apoptosis. Clostridial cytotoxins specifically inactivate GTPases by glucosylation [ Clostridium difficile toxin B-10463 (TcdB-10463), C. difficile toxin B-1470 (TcdB-1470)] or ADP ribosylation ( C. botulinum C3 toxin). Exposure of human umbilical cord vein endothelial cells (HUVEC) to TcdB-10463, which inhibits RhoA/Rac1/Cdc42, or to C3 toxin, which inhibits RhoA, -B, -C, resulted in apoptosis, whereas inactivation of Rac1/Cdc42 with TcdB-1470 was without effect, suggesting that Rho inhibition was responsible for endothelial apoptosis. Disruption of endothelial microfilaments as well as inhibition of p160ROCK did not induce endothelial apoptosis. Exposure to TcdB-10463 resulted in activation of caspase-9 and -3 but not caspase-8 in HUVEC. Moreover, Rho inhibition reduced expression of antiapoptotic Bcl-2 and Mcl-1 and increased proapoptotic Bid but had no effect on Bax or FLIP protein levels. Caspase-3 activity and apoptosis induced by TcdB-10463 were abolished by cAMP elevation. In summary, inhibition of Rho in endothelial cells activates caspase-9- and -3-dependent apoptosis, which can be antagonized by cAMP elevation.
In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages
Authors: Poelma D. L. H. , Zimmermann L. J. I. , Scholten H. H. , Lachmann B. , van Iwaarden J. F.
Published: March 2015

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70–75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.
Vascular segmental permeabilities at high peak inflation pressure in isolated rat lungs
Authors: Parker J. C. , Yoshikawa S.
Published: March 2015

The response of segmental filtration coefficients ( K f) to high peak inflation pressure (PIP) injury was determined in isolated perfused rat lungs. Total ( K f,t), arterial ( K f,a), and venous ( K f,v) filtration coefficients were measured under baseline conditions and after ventilation with 40–45 cmH2O PIP. K f,a and K f,v were measured under zone I conditions by increasing airway pressure to 25–27 cmH2O. The microvascular segment K f ( K f,mv) was then calculated by: K f,mv = K f,t − K f,a − K f,v. The baseline K f,twas 0.090 ± 0.022 ml · min−1 · cmH2O−1 · 100 g−1 and segmentally distributed 18% arterial, 41% venous, and 41% microvascular. After high PIP injury, K f,t increased by 680%, whereas K f,a, K f,v, and K f,mv increased by 398, 589, and 975%, respectively. Pretreatment with 50 μM gadolinium chloride prevented the high PIP-induced increase in K f in all vascular segments. These data imply a lower hydraulic conductance for microvascular endothelium due to its large surface area and a gadolinium-sensitive high-PIP injury, produced in both alveolar and extra-alveolar vessel segments.
Inhibition of compensatory lung growth in endothelial nitric oxide synthase-deficient mice
Authors: Leuwerke Shari M. , Kaza Aditya K. , Tribble Curtis G. , Kron Irving L. , Laubach Victor E.
Published: March 2015

Pneumonectomy results in rapid compensatory growth of the remaining lung and also leads to increased flow and shear stress, which are known to stimulate endothelial nitric oxide synthase (eNOS). Nitric oxide is an essential mediator of vascular endothelial growth factor-induced angiogenesis, which should necessarily occur during compensatory lung growth. Thus our hypothesis is that eNOS is critical for compensatory lung growth. To test this, left pneumonectomy was performed in eNOS-deficient mice (eNOS−/−), and compensatory growth of the right lung was characterized throughout 14 days postpneumonectomy and compared with wild-type pneumonectomy and sham controls. Compensatory lung growth was severely impaired in eNOS−/− mice, as demonstrated by significant reductions in lung weight index, lung volume index, and volume of respiratory region. Also, pneumonectomy-induced increases in alveolar surface density and cell proliferation were prevented in eNOS−/− mice, indicating that eNOS plays a role in alveolar hyperplasia. Compensatory lung growth was also impaired in wild-type mice treated with the nitric oxide synthase inhibitor N G-nitro-l-arginine methyl ester. Together, these results indicate that eNOS is critical for compensatory lung growth.
Role of heme oxygenases in sepsis-induced diaphragmatic contractile dysfunction and oxidative stress
Authors: Barreiro Esther , Comtois Alain S. , Mohammed Shawn , Lands Larry C. , Hussain Sabah N. A.
Published: March 2015

Heme oxygenases (HOs), essential enzymes for heme metabolism, play an important role in the defense against oxidative stress. In this study, we evaluated the expression and functional significance of HO-1 and HO-2 in the ventilatory muscles of normal rats and rats injected with bacterial lipopolysaccharide (LPS). Both HO-1 and HO-2 proteins were detected inside ventilatory and limb muscle fibers of normal rats. Diaphragmatic HO-1 and HO-2 expressions rose significantly within 1 and 12 h of LPS injection, respectively. Inhibition of the activity of inducible nitric oxide synthase (iNOS) in rats and absence of this isoform in iNOS−/− mice did alter sepsis-induced regulation of muscle HOs. Systemic inhibition of HO activity with chromium mesoporphyrin IX enhanced muscle protein oxidation and hydroxynonenal formation in both normal and septic rats. Moreover, in vitro diaphragmatic force generation declined substantially in response to HO inhibition both in normal and septic rats. We conclude that both HO-1 and HO-2 proteins play an important role in the regulation of muscle contractility and in the defense against sepsis-induced oxidative stress.
Coupling of sympathetic nerve traffic and BP at very low frequencies is mediated by large-amplitude events
Authors: Burgess Don E. , Randall David C. , Speakman Richard O. , Brown David R.
Published: March 2015

This study explores the functional association between renal sympathetic nerve traffic (NT) and arterial blood pressure (BP) in the very-low-frequency range (i.e., <0.1 Hz). NT and BP ( n = 6) or BP alone ( n = 17) was recorded in unanesthetized rats ( n = 6). Data were collected for 2–5 h, and wavelet transforms were calculated from data epochs of up to 1 h. From these transforms, we obtained probability distributions for fluctuation amplitudes over a range of time scales. We also computed the cross-wavelet power spectrum between NT and BP to detect the occurrence in time of large-amplitude transient events that may be important in the autonomic regulation of BP. Finally, we computed a time sequence of cross correlations between NT and BP to follow the relationship between NT and BP in time. We found that NT and BP follow comparable self-similar scaling relationships (i.e., NT and BP fluctuations exhibit a certain type of power law behavior). Scaling of this nature 1) points to underlying dynamics over a wide range of scales and 2) is related to large-amplitude events that contribute to the very-low-frequency variability of NT and BP. There is a strong correlation between NT and BP during many of these transient events. These strong correlations and the uniformity in scaling imply a functional connection between these two signals at frequencies where we previously found no connection using spectral coherence.
Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice
Authors: Pitts SiNae , Perone Elizabeth , Silver Rae
Published: March 2015

Daily scheduled feeding is a potent time cue that elicits anticipatory activity in rodents. This food-anticipatory activity (FAA) is controlled by a food-entrainable oscillator (FEO) that is distinct from light-entrained oscillators of the suprachiasmatic nucleus (SCN). Circadian rhythms within the SCN depend on transcription-translation feedback loops in which CLOCK protein is a key positive regulator. The Clock gene is expressed in rhythmic tissues throughout the brain and periphery, implicating its widespread involvement in the functioning of circadian oscillators. To examine whether CLOCK protein is also necessary for the FEO, the effect of daily food restriction was studied in homozygous Clock mutant ( Clk/Clk) mice. The results show that Clk/Clk mutant mice exhibit FAA, even when their circadian wheel-running behavior is arrhythmic. As in wild-type controls, FAA in Clk/Clk mutants persists after temporal feeding cues are removed for several cycles, indicating that the FEO is a circadian timer. This is the first demonstration that the Clock gene is not necessary for the expression of a circadian, food-entrained behavior and suggests that the FEO is mediated by a molecular mechanism distinct from that of the SCN.
Increased connexin43-mediated intercellular communication in a rat model of bladder overactivity in vivo
Authors: Christ George J. , Day Nancy S. , Day Michele , Zhao Weixin , Persson Katarina , Pandita Raj K. , Andersson Karl-Erik
Published: March 2015

Bladder overactivity associated with outflow obstruction is a common human condition recapitulated in the female rat by narrowing the diameter of the urethra. The goal of these studies was to evaluate the role of intercellular communication through connexin43 (Cx43)-derived gap junction channels to bladder overactivity following partial urethral outflow obstruction of 3-day to 6-wk duration. Cx43 mRNA and protein expression were barely detectable by Northern or Western blots, respectively, in the detrusor layer of normal bladders, but bands were found with both techniques after 6 wk of obstruction. Linear regression analysis of the RT-PCR data revealed a statistically significant positive correlation between the duration of obstruction (again, ranging from 3-day to 6-wk duration) and Cx43 mRNA transcript levels, such that after 6 wk of obstruction, Cx43 transcript levels were ≈15-fold greater than initial control values. When taking into account the approximately fivefold increase in bladder weight over this same time frame, the absolute amount of Cx43 mRNA in the bladder apparently increased by ≈75-fold. In that regard, as anticipated, and consistent with previous observations, 6 wk of obstruction was also associated with a significant increase in spontaneous bladder contractions between micturitions. The amplitude of these contractions was significantly reduced by heptanol given intravesically. Furthermore, carbachol-precontracted bladder strips from obstructed animals were more sensitive to heptanol-induced relaxation (100 μM) than their unobstructed counterparts ( n = 6; P < 0.01). When bladder strips were equivalently precontracted via electrical field stimulation (EFS; 20 Hz), similar heptanol-induced relaxation responses were observed. However, the tetrodotoxin-resistant portion of the EFS-induced contraction was greater in the obstructed than in the unobstructed animals, and this portion of the contractile response was more sensitive to heptanol-induced relaxation in obstructed than unobstructed bladders ( n = 7; P < 0.01). Taken together, these observations indicate that partial outlet obstruction produces an overactive bladder that may be more dependent on intercellular communication through gap junctions for modulation of contractile responses than its normal counterpart.
Evidence for microvascular dysfunction after prenatal dexamethasone at 0.7, 0.75, and 0.8 gestation in sheep
Authors: Molnar Judit , Nijland Mark J. M. , Howe David C. , Nathanielsz Peter W.
Published: March 2015

Dexamethasone (DM) was administered to pregnant ewes as three weekly courses of four injections of 2 mg at 12-h intervals. DM ( n = 7) or saline ( n= 7) was given starting at 103 days of gestation (dGA; term ∼149 days). Fetal femoral arteries (∼300-μm internal diameter) were evaluated using wire myography at 119 dGA. DM-exposed fetuses were significantly smaller than saline-exposed fetuses. DM exposure increased maximal contraction to 125 mM KCl, and maximum tension developed along with sensitivity to endothelin-1 and relaxation to bradykinin. Preincubation with the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester shifted the dose-response curves to endothelin-1 and acetylcholine to the right in controls but not in the DM-exposed group. Relaxation to acetylcholine and to the nitric oxide donor sodium nitroprusside was similar in both groups. The combination of enhanced endothelin-induced vasoconstriction, abnormal endothelium-dependent relaxation, and normal endothelium-independent relaxation indicates microvessel dysfunction following antenatal DM administration. Because such dysfunction is associated with several forms of adult hypertension, our results indicate the potential for consequences of antenatal glucocorticoid exposure on adult cardiovascular health.
Importance of satellite cells in the strength recovery after eccentric contraction-induced muscle injury
Authors: Rathbone Christopher R. , Wenke J. C. , Warren Gordon L. , Armstrong R. B.
Published: March 2015

The purpose of this study was to determine if the elimination of satellite cell proliferation using γ-irradiation would inhibit normal force recovery after eccentric contraction-induced muscle injury. Adult female ICR mice were implanted with a stimulating nerve cuff on the common peroneal nerve and assigned to one of four groups: 1) irradiation- and eccentric contraction-induced injury, 2) eccentric contraction-induced injury only, 3) irradiation only, and 4) no intervention. Anterior crural muscles were irradiated with a dose of 2,500 rad and injured with 150 in vivo maximal eccentric contractions. Maximal isometric torque was determined weekly through 35 days postinjury. Immediately after injury, maximal isometric torque was reduced by ∼50% and had returned to normal by 28 days postinjury in the nonirradiated injured mice. However, torque production of irradiated injured animals did not recover fully and was 25% less than that of injured nonirradiated mice 35 days postinjury. These data suggest that satellite cell proliferation is required for approximately half of the force recovery after eccentric contraction-induced injury.
NO mediates effects of estrogen on central regulation of blood pressure in restrained, ovariectomized rats
Authors: Cherney Anton , Edgell Heather , Krukoff Teresa L.
Published: March 2015

We tested the hypotheses that estrogen replacement in ovariectomized (OVX) rats attenuates cardiovascular responses to psychological stress and that nitric oxide (NO) in the brain mediates these effects. Female rats were OVX; one group received 17β-estradiol (OVX-E) for 11-12 days and the other received vehicle (OVX-V). Seven days after OVX, OVX-E and OVX-V rats were chronically instrumented for arterial pressure measurements and intracerebroventricular injections. Later (4-5 days), OVX-E and OVX-V rats received intracerebroventricular injections of NG-nitro-l-arginine (88 μg/kg), an inhibitor of constitutive NO production, or vehicle. Mean arterial pressure (MAP) and heart rate responses were then measured in conscious rats exposed to two cycles of 1-h restraint/1-h rest. We show that MAP responses in restrained OVX-E rats were attenuated both during restraint and during rest. Although inhibition of NO production in the brain had no effect on MAP responses to restraint in OVX-V rats, it augmented responses in restrained OVX-E rats, especially during periods of rest, so that MAPs in restrained OVX-E and OVX-V rats were indistinguishable. Finally, NO levels in hypothalami and brain stems were elevated in restrained OVX-E, but not OVX-V, rats compared with their respective unrestrained controls. These results show that estrogen replacement in OVX rats reduces arterial pressure responses to psychological stress and that these effects are mediated, at least in part, by NO.
Naltrexone infusion inhibits the development of preference for a high-sucrose diet
Authors: Levine Allen S. , Grace Martha K. , Cleary James P. , Billington Charles J.
Published: March 2015

We hypothesized that the opioid antagonist naltrexone would inhibit the redevelopment of a preference for a high-sucrose diet after an abstention period from this diet. Rats that chose between a starch or sucrose diet for 10 days preferred the sucrose diet. Rats were then given access to the starch diet alone for another 10-day period. A miniosmotic pump containing saline or naltrexone was then implanted (70 μg/h; 1.7 mg/day) for ∼10 days. During the saline infusion, 77% of the total energy came from the sucrose diet, whereas during the naltrexone infusion, 33% of the total energy came from the sucrose diet. We repeated this study in another group of rats but did not restrict the sucrose diet. In this case naltrexone failed to decrease preference for the sucrose diet. Thus naltrexone infusion inhibited redevelopment of a preference for a sucrose diet after a period of restriction to a starch diet for 10 days but had no effect on preference if both diets were present throughout the study.
Chemical activation of C1–C2 spinal neurons modulates activity of thoracic respiratory interneurons in rats
Authors: Qin C. , Farber J. P. , Chandler M. J. , Foreman R. D.
Published: March 2015

Discharge patterns of thoracic dorsal horn neurons are influenced by chemical activation of cell bodies in cervical spinal segments C1–C2. The present aim was to examine whether such activation would specifically affect thoracic respiratory interneurons (TRINs) of the deep dorsal horn and intermediate zone in pentobarbital sodium-anesthetized, paralyzed, artificially ventilated rats. We also characterized discharge patterns and pathways of TRIN activation in rats. A total of 77 cells were classified as TRINs by location, continued burst activity related to phrenic discharge when the respirator was stopped, and lack of antidromic response from selected pathways. A variety of respiration-phased discharge patterns was documented whose pathways were interrupted by ipsilateral C1 transection. Glutamate pledgets (1 M, 1 min) on the dorsal surface of the spinal cord inhibited 22/49, excited 15/49, or excited/inhibited 3/49 tested cells. Incidence of responses did not depend on whether the phase of TRIN discharge was inspiratory, expiratory, or biphasic. Phrenic nerve activity was unaffected by chemical activation of C1–C2 in this preparation. Besides supraspinal input, TRIN activity may be influenced by upper cervical modulatory pathways.
Stress facilitates body weight gain in genetically predisposed rats on medium-fat diet
Authors: Michel Chantal , Levin Barry E. , Dunn-Meynell Ambrose A.
Published: March 2015

To assess the interaction between stress and energy homeostasis, we immobilized male Sprague-Dawley rats prone to diet-induced obesity (DIO) or diet resistance (DR) once for 20 min and then fed them either low-fat (4.5%) chow or a medium-fat (31%), high-energy (HE) diet for 9 days. Stressed, chow-fed DIO rats gained less, while stressed DIO rats on HE diet gained more body weight and had higher feed efficiency and plasma leptin levels than unstressed controls. Neither stress nor diet affected DR body weight gain. While stress-induced plasma corticosterone levels did not differ between phenotypes, DIO rats were initially more active in an open field and had higher hippocampal dentate gyrus and CA1 glucocorticoid receptor (GR) mRNA than DR rats, regardless of prior stress or diet. HE diet intake was associated with raised dentate gyrus and CA1 GR and amygdalar central nucleus (CeA) corticotropin-releasing hormone (CRH) mRNA expression, while stress was associated with reduced hypothalamic dorsomedial nucleus Ob-R mRNA and CeA CRH specifically in DIO rats fed HE diet. Thus a single stress triggers a complex interaction among weight gain phenotype, diet, and stress responsivity, which determines the body weight and adiposity of a given individual.
Renal endothelin in chronic angiotensin II hypertension
Authors: Sasser Jennifer M. , Pollock Jennifer S. , Pollock David M.
Published: March 2015

To determine the influence of chronic ANG II infusion on urinary, plasma, and renal tissue levels of immunoreactive endothelin (ET), ANG II (65 ng/min) or saline vehicle was delivered via osmotic minipump in male Sprague-Dawley rats given either a high-salt diet (10% NaCl) or normal-salt diet (0.8% NaCl). High-salt diet alone caused a slight but not statistically significant increase (7 ± 1%) in mean arterial pressure (MAP). MAP was significantly increased in ANG II-infused rats (41 ± 10%), and the increase in MAP was significantly greater in ANG II rats given a high-salt diet (59 ± 1%) compared with the increase observed in rats given a high-salt diet alone or ANG II infusion and normal-salt diet. After a 2-wk treatment, urinary excretion of immunoreactive ET was significantly increased by ∼50% in ANG II-infused animals and by over 250% in rats on high-salt diet, with or without ANG II infusion. ANG II infusion combined with high-salt diet significantly increased immunoreactive ET content in the cortex and outer medulla, but this effect was not observed in other groups. In contrast, high-salt diet, with or without ANG II infusion, significantly decreased immunoreactive ET content within the inner medulla. These data indicate that chronic elevations in ANG II levels and sodium intake differentially affect ET levels within the kidney and provide further support for the hypothesis that the hypertensive effects of ANG II may be due to interaction with the renal ET system.
Glucose regulates lipid metabolism in fasting king penguins
Authors: Bernard Servane F. , Orvoine Jord , Groscolas René
Published: March 2015

This study aims to determine whether glucose intervenes in the regulation of lipid metabolism in long-term fasting birds, using the king penguin as an animal model. Changes in the plasma concentration of various metabolites and hormones, and in lipolytic fluxes as determined by continuous infusion of [2-3H]glycerol and [1-14C]palmitate, were examined in vivo before, during, and after a 2-h glucose infusion under field conditions. All the birds were in the phase II fasting status (large fat stores, protein sparing) but differed by their metabolic and hormonal statuses, being either nonstressed (NSB; n = 5) or stressed (SB; n = 5). In both groups, glucose infusion at 5 mg·kg-1·min-1 induced a twofold increase in glycemia. In NSB, glucose had no effect on lipolysis (maintenance of plasma concentrations and rates of appearance of glycerol and nonesterified fatty acids) and no effect on the plasma concentrations of triacylglycerols (TAG), glucagon, insulin, or corticosterone. However, it limited fatty acid (FA) oxidation, as indicated by a 25% decrease in the plasma level of β-hydroxybutyrate (β-OHB). In SB, glucose infusion induced an ∼2.5-fold decrease in lipolytic fluxes and a large decrease in FA oxidation, as reflected by a 64% decrease in the plasma concentration of β-OHB. There were also a 35% decrease in plasma TAG, a 6.5- and 2.8-fold decrease in plasma glucagon and corticosterone, respectively, and a threefold increase in insulinemia. These data show that in fasting king penguins, glucose regulates lipid metabolism (inhibition of lipolysis and/or of FA oxidation) and affects hormonal status differently in stressed vs. nonstressed individuals. The results also suggest that in birds, as in humans, the availability of glucose, not of FA, is an important determinant of the substrate mix (glucose vs. FA) that is oxidized for energy production.
Increased thirst and vasopressin secretion after myocardial infarction in rats
Authors: De Smet H. R. , Menadue M. F. , Oliver J. R. , Phillips P. A.
Published: March 2015

Impaired regulation of salt and water balance in left ventricular dysfunction and heart failure can lead to pulmonary and peripheral edema and hyponatremia. Previous studies of disordered water regulation in heart failure have used models of low cardiac output with normal cardiac function (e.g., inferior vena cava ligation). We investigated thirst and vasopressin (AVP) secretion in a rat myocardial infarction model of chronic left ventricular dysfunction/heart failure in response to a 24-h water deprivation period. Thirst (implied from water drunk), hematocrit, plasma renin activity, and plasma AVP concentrations increased with water deprivation vs. ad libitum water access. Thirst and plasma AVP concentrations were significantly positively correlated with infarct size after 24-h water deprivation but not under ad libitum water access conditions. The mechanism by which this occurs is unclear but could involve increased osmoreceptor sensitivity, altered stimulation of baroreceptors, the renin-angiotensin system, or altered central neural control.
Effect of NOS inhibition on central response to atrial distension during pregnancy
Authors: Tam Siu Lin , Sims Elaine , Kaufman Susan
Published: March 2015

Atrial distension increases c- fosexpression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either d- or N G-nitro-l-arginine methyl ester (l-NAME) (120 mg/2 ml at 10 μg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c- fos expression. In the virgin rats, and in the pregnant rats treated with l-NAME, atrial distension significantly increased hypothalamic c- fosexpression. In the pregnant animals treated with d-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.