Subunit-specific coordinate upregulation of sodium pump activity in alveolar epithelial cells by lentivirus-mediated gene transfer

Resolution of alveolar edema depends on active ion transport by sodium pumps located on the basolateral surface of alveolar epithelial cells (AECs), suggesting that upregulation of sodium pump activity may facilitate clearance of edema fluid. We have investigated the use of lentiviral vectors to augment sodium pump activity via gene transfer of sodium pump subunits to AECs. Full-length cDNA for the alpha(1) or beta(1) subunit of rat Na(+),K(+)-ATPase was cloned into the lentiviral vector pRRLsin.hCMV.IRES.EGFP. Rat AECs in primary culture were transduced on day 4 with lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G. Transduction with lentiviral vectors encoding either alpha(1) subunit (Lenti-alpha(1)-EGFP) or beta(1) subunit (Lenti-beta(1)-EGFP) led to dose-dependent increases in mRNA and protein for the corresponding subunit. Transduction with Lenti-beta(1)-EGFP was accompanied by coordinate upregulation of endogenous alpha(1) expression, whereas endogenous beta(1) expression was unchanged after transduction with Lenti-alpha(1)-EGFP. Consistent with these findings, transduction with Lenti-beta(1)-EGFP, but not Lenti-alpha(1)-EGFP, led to augmentation of sodium pump activity as a result of increases in Na(+),K(+)-ATPase holoenzyme. Sodium pump alpha(2) subunit and sodium channel protein did not change after Lenti-beta(1)-EGFP transduction. These results demonstrate that overall sodium pump activity can be efficiently upregulated in AECs specifically via gene transfer of the sodium pump beta(1) subunit and support the feasibility of lentivirus-mediated gene transfer to augment alveolar fluid clearance.     

T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase

Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na(+)/K(+)-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1(-/-)), but not IFN-gamma knockout mice. Anti-CD3 mAb decreased mucosal Na(+)/K(+)-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1(-/-) mice. Neither alpha nor beta subunits of Na(+)/K(+)-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3-treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na(+) absorption, Na(+)-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR(-/-) mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na(+)/K(+)-ATPase activity leading to decreased intestinal Na(+) and water absorption.     

Effects of maternal diabetes on fetal rat lung ion transport. Contribution of alveolar and bronchiolar epithelial cells to Na+,K(+)-ATPase expression.

Fetuses of streptozotocin-induced diabetic rats exhibited delayed lung maturation and a 40% reduction in the steady-state level of lung Na+,K(+)-ATPase alpha 1 subunit mRNA and Na+,K(+)-ATPase activity at 21 d of gestation. In in situ hybridization experiments the signal specific for Na(+)-pump alpha 1 subunit message was strongest above columnar epithelial cells of air-conducting structures. Strong labeling was also present above cuboidal cells lining the forming alveoli, but not above mesenchymal cells. Immunocytochemical localization of the protein paralleled the distribution of the mRNA. Mesenchymal cells were more abundant in fetal lungs of diabetic mothers, and thus the decreased overall levels of Na+,K(+)-ATPase may result from the observed morphological pulmonary immaturity. One day after birth there was no apparent difference in lung morphology at the light microscopic level, in the localization or the steady-state level of Na+,K(+)-ATPase alpha 1 isoform mRNA, or in enzyme activity. Na+,K(+)-ATPase has a likely role in the active phase of fluid absorption in the airways of newborns before the onset of breathing. Decreased fluid clearance and lack of thinning of the lung's connective tissue may contribute to the increased risk for respiratory distress in infants of diabetic mothers.     

Inhibition of Na(+)/K(+)-atpase by endothelin-1 in human nonpigmented ciliary epithelial cells

Endothelin-1 (ET-1), a potent vasoconstrictor, lowers intraocular pressure in mammals, either by enhancing the outflow of aqueous humor (AH) via the trabecular meshwork and Schlemm's canal or by reducing AH formation at the ciliary epithelium. Aqueous humor production occurs by passive diffusion of water coupled with active transport of ions, mainly involving Na(+):K(+):2Cl(-) cotransporter and Na(+)/K(+)-ATPase pump from serosal to aqueous side. Presently, we have evaluated the effects of ET-1 on Na(+):K(+):2Cl(-) cotransport and Na(+)/K(+)-ATPase activity in HNPE cells using (86)Rb(+) uptake. ET-1 (100 pM-100 nM) decreased mean (86)Rb(+) uptake by 15% during a 15-min uptake period. ET-1's effect was not prevented by BQ610, an ET(A) receptor antagonist, but was blocked by BQ788, an ET(B) receptor antagonist. ET-1's effect was mimicked by sarafotoxin, an ET(B) agonist. ET-1-induced reduction in (86)Rb(+) uptake was additive with bumetanide, a selective inhibitor of Na(+):K(+):2Cl(-) cotransporter but not with ouabain, a selective inhibitor of the Na(+)/K(+)-ATPase. ET-1 did not affect iberiotoxin-sensitive maxi K(+) channels. This suggests that ET-1-induced reduction in (86)Rb(+) uptake is mediated through the inhibition of the Na(+)/K(+)-ATPase via an ET(B)-like receptor. These findings are consistent with an ET-1 effect on active ion transport activity in HNPE cells that could explain the reduction in aqueous humor production and the lowering of intraocular pressure.     

OuaSelect, a novel ouabain-resistant human marker gene that allows efficient cell selection within 48 h

Efficient selection of gene-modified cells is required for a number of potential gene therapy applications, as well as molecular biology studies. Ideally, a clinical selection regimen would combine high selection speed, efficiency and efficacy, in addition to clinical grade selection techniques and low immunogenicity. To our knowledge, a selection marker satisfying all these features is so far not available. Ouabain is a clinically used cardiac glycoside and selective Na(+)/K(+)-ATPase inhibitor. On the basis of the high sensitivity of human Na(+)/K(+)-ATPase proteins to ouabain, and rapid killing of cells upon exposure, we have screened the ubiquitously expressed Na(+)/K(+)-ATPase alpha1 subunit for mutations that could greatly increase its resistance to ouabain. Two amino-acid substitutions, Q118R and N129D were sufficient to confer a two log greater resistance to ouabain in HeLa, Jurkat, U2OS cells and in primary cells. Furthermore, following transduction of primary lymphocytes with the alpha1(Q118R/N129D) gene, >99% pure populations of gene-modified cells were achieved with a recovery rate of >80% after 48 h of exposure to ouabain. These results identify the human alpha1(Q118R/N129D) (OuaSelect) as a promising selection marker gene for safe, rapid and cost-effective selection in clinical gene therapy and molecular biology research.     

Expression and developmental regulation of Na+,K+ adenosine triphosphatase in the rat small intestine.

The Na+,K(+)-ATPase ion pump plays a critical role in fluid and electrolyte physiology of the small intestine. Here we show that, of the three known alpha isotypes (alpha 1, alpha 2, and alpha 3) of the sodium pump found in the rat, only alpha 1 is expressed in the small intestine. The expression of this isotype, considered at the level of mRNA, is under developmental control, with the adult intestine exhibiting approximately a threefold increase in alpha 1 message over the neonate. Cortisone treatment of the neonate results in near-adult levels of alpha 1 mRNA expression. An increase in the abundance of alpha 1 isotype parallels the changes in its mRNA expression. beta subunit mRNA is expressed coordinately with the alpha 1 subunit mRNA. A four- to five-fold rise in the Na+,K(+)-ATPase activity is also developmentally induced.     

Na(+)/K(+)-ATPase inhibition upregulates NMDA-evoked currents in rat hippocampal CA1 pyramidal neurons

Na(+)/K(+)-ATPase and N-methyl-D-aspartate (NMDA) receptor in hippocampus play very important roles in the regulation of learning and memory. Here, we showed that dihydroouabain (DHO, 10(-5)-10(-3) M), a Na(+)/K(+)-ATPase inhibitor, significantly potentiated NMDA current in rat hippocampal CA1 pyramidal neurons, which was blocked by PP2 (the selective Src tyrosine kinase inhibitor) and PD-98059 [the selective inhibitor of the mitogen-activated protein kinases (MAPK) cascade]. These findings reported here uncover that Src mediates the cross-talk between Na(+)/K(+)-ATPase and NMDA receptor to transduce the signals from Na(+)/K(+)-ATPase to the MAPK cascade and provide new insights into therapeutic target for deeper understanding of the nature of cognitive disorder.     

Lack of effect of clinical doses of cyclosporin A on erythrocyte Na+/K+-ATPase activity.

Cyclosporin A (CsA) may exert its cytotoxic effects by altering the activity of different plasma membrane transport systems. Although CsA may act at the gene level, it has been also suggested that it can directly alter transport processes at the plasma membrane. To examine this possibility in a physiological context, we determined Na(+)/K(+)-ATPase activity in erythrocytes from two groups of subjects receiving CsA treatment: group I consisted of kidney transplant patients, and group II comprised patients with steroid-resistant idiopathic nephrotic syndrome. Group I patients showed a marked decrease (35%) in the activity of the Na(+)/K(+)-ATPase in erythrocytes immediately after surgery, before the initiation of CsA treatment. The activity remained low 2 days after the introduction of CsA, but had recovered to the original (pre-surgery) value 1 month later. Group II patients showed the same pattern of erythrocyte Na(+)/K(+)-ATPase activity as those in group I. When the blood CsA levels from all patients were plotted against the corresponding erythrocyte Na(+)/K(+)-ATPase transport activity, a significant linear correlation was found. Higher levels of CsA in the blood were correlated significantly with increased Na(+)/K(+)-ATPase activities. The blood sodium concentration was also correlated positively with both erythrocyte Na(+)/K(+)-ATPase activity and blood CsA concentration. Thus CsA treatment is not associated with inhibition of the Na(+)/K(+)-ATPase in erythrocytes.     

Impairment of sodium pump and Na/H exchanger in erythrocytes from non-insulin dependent diabetes mellitus patients: effect of tea catechins

BACKGROUND: Tea catechins (EGCG, EGC, ECG and EC) possess many important biological properties. We evaluated the effect of tea catechins on erythrocyte membrane Na(+)/K(+)-ATPase and sodium/hydrogen exchanger (NHE) activity in normal (control) and NIDDM subjects. METHODS: Erythrocyte membrane Na(+)/K(+)-ATPase and NHE activity were determined in normal and non-insulin dependent diabetes mellitus (NIDDM) patients. In vitro effect of tea catechins was studied by incubating membrane/intact erythrocytes in assay medium prior to Na(+)/K(+)-ATPase/NHE activity determination. RESULTS: A 24.2% decrease in the activity of Na(+)/K(+)-ATPase (p<0.001) and 39.37% increase in activity of NHE (p<0.02) were observed in NIDDM subjects compared to normal. Tea catechins inhibited the activity of Na(+)/K(+)-ATPase and NHE in both normal and NIDDM erythrocytes, the effect was concentration-dependent. The inhibitory effect of EGCG and ECG at micromolar concentrations was greater compared to EGC and EC on Na(+)/K(+)-ATPase. On NHE the inhibition of tea catechins was in the order: EC>EGC>ECG>EGCG at concentrations up to 10 micromol/l. CONCLUSIONS: This data may help to explain the anti-carcinogenic and cardioprotective effects of tea catechins. The effect of tea catechins on Na(+)/K(+)-ATPase and NHE may be explained due to a direct effect of these compounds on plasma membrane leading to a change in membrane fluidity.     

Presence of a functional TSH receptor on human erythrocytes.

The presence of thyrotropin receptors (TSHR) has been reported in some extrathyroidal tissues but its physio-pathological role still remains unclear. TSH (seems to) affects the human erythrocytes Na(+)/K(+)-ATPase in vitro, however receptors on erythrocytes have not yet been described. In this work the effect of recombinant human TSH (rhTSH) on sites number and activity of erythrocyte Na(+)/K(+)-ATPase was investigated in a group of thyroidectomized patient enrolled for rhTSH test. As detected by (3)H-ouabain binding, rhTSH administration induced a significant increase in the number of sites (p=0.005) and in the Kd (p=0.006) of Na(+)/K(+)-ATPase. rhTSH did not induce significant difference in Na(+)/K(+)-ATPase activity measured by (86)Rb uptake. (125)I-TSH binding studies and Western blotting data showed the existence of TSHR in the erythrocytes of healthy donors. In conclusion The TSH action on Na(+)/K(+)-ATPase of human erythrocytes can be explained by the presence of TSHR.     

Inhibition of lipid peroxidation, protein glycation and elevation of membrane ion pump activity by taurine in RBC exposed to high glucose

BACKGROUND: Supplementation of taurine, a sulfur containing amino acid has been found to be beneficial in counteracting oxidative stress and in preventing experimental diabetic neuropathy, nephropathy and retinopathy. Taurine has its own capacity to prevent the suppression of membrane-bound Na(+)/K(+)ATPase activity and prevent Ca(2+) overload. This study was undertaken to test whether taurine can reduce lipid peroxidation and glycosylation and can increase the Na(+)/K(+)- and Ca(2+)-ATPase activities in high glucose-treated red blood cells (RBC). METHODS: Washed normal human RBC were incubated in phosphate-buffered saline with normal (6 mmol/l) or high glucose concentrations (45 mmol/l), with and without 50-150 micromol/l taurine in a shaking water bath at 37 degrees C for 24 h. Lipid peroxidation, glycated hemoglobin, glucose utilization and Na(+)/K(+)- and Ca(2+)-ATPase activities were determined in the glucose-treated human RBC. RESULTS: Taurine significantly lowered the level of glycated hemoglobin (GHb) and lipid peroxidation in RBC exposed to high glucose concentrations. Stimulation of glucose utilization by RBC was significant in the presence of taurine both in normal and high glucose-treated RBC. The activities of Na(+)/K(+)- and Ca(2+)-ATPases in RBC membranes were significantly lowered in high glucose-treated RBC. Taurine treatment significantly prevented the reduction in activities of Na(+)/K(+)- and Ca(2+)-ATPases activities in high glucose-treated RBC. CONCLUSIONS: The results show that taurine is important for the physiological functions of RBCs and the effects of taurine on glucose-treated RBC may have potential therapeutic relevance in diabetes.     

Increased reabsorption of alveolar edema fluid in the obese Zucker rat

Diabetic patients have a decreased incidence of acute respiratory distress syndrome, but the mechanism responsible for the decreased incidence is uncertain. Reabsorption of alveolar edema fluid (alveolar fluid clearance) has been considered to play an important role in resolution of acute respiratory distress syndrome. However, little is known regarding alveolar fluid clearance in diabetes mellitus. Since the obese Zucker rat has been used as an experimental model for diabetes mellitus, we determined if alveolar fluid clearance increased in the obese Zucker rat. First, we compared alveolar fluid clearance in obese Zucker rats with that in lean Zucker rats and Sprague-Dawley (SD) rats. Then, we determined the role of sodium channel, Na,K-ATPase, and beta(2)-adrenoceptor, which drives alveolar fluid clearance, in obese Zucker rats. Alveolar fluid clearance was estimated by the progressive increase in alveolar albumin concentrations in the isolated lungs. We found that basal alveolar fluid clearance in obese Zucker rats was two-fold greater than that in lean Zucker rats and SD rats. The mRNA expression of alpha(1)-, beta(1)-Na, K-ATPase and beta(2)-adrenoceptor, but not mRNA expression of sodium channel, increased in obese Zucker rats. A selective beta(2)-agrenergic antagonist, but not a Na, K-ATPase inhibitor, specifically inhibited the increase in alveolar fluid clearance in obese Zucker rats. These results indicate that overexpression of beta(2)-adrenoceptor primarily increases basal alveolar fluid clearance in the obese Zucker rat. We speculate that the stimulation of alveolar fluid clearance ameliorates acute respiratory distress syndrome in patients with diabetes mellitus.     

Pretreatment with cationic lipid-mediated transfer of the Na+K+-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema

Resolution of pulmonary oedema is mediated by active absorption of liquid across the alveolar epithelium. A key component of this process is the sodium-potassium ATPase (Na+K+-ATPase) enzyme located on the basolateral surface of epithelial cells and up-regulated during oedema resolution. We hypothesised that lung liquid clearance could be further up-regulated by lipid-mediated transfer and expression of exogenous Na+K+-ATPase cDNA. We demonstrate proof of this principle in a model of high permeability pulmonary oedema induced by intraperitoneal injection of thiourea (2.5 mg/kg) in C57/BL6 mice. Pretreatment of mice (24 h before thiourea) by nasal sniffing of cationic liposome (lipid #67)-DNA complexes encoding the alpha and beta subunits of Na+K+-ATPase (160 microg per mouse), significantly (P<0.01) decreased the wet:dry weight ratios measured 2 h after thiourea injection compared with control animals, pretreated with an equivalent dose of an irrelevant gene. Whole lung Na+K+-ATPase activity was significantly (P<0.05) increased in mice pretreated with Na+K+-ATPase cDNA compared both with untreated control animals as well as animals pretreated with the irrelevant gene. Nested RT-PCR on whole lung homogenates confirmed gene transfer by detection of vector-specific mRNA in three of four mice studied 24 h after gene transfer. This demonstration of a significant reduction in pulmonary oedema following in vivo gene transfer raises the possibility of gene therapy as a novel, localised approach for pulmonary oedema in clinical settings such as ARDS and lung transplantation.     

20-Hydroxyeicosa-tetraenoic acid (20 HETE) activates protein kinase C. Role in regulation of rat renal Na+,K+-ATPase.

It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.     

肺泡内液体清除与肺水肿的研究进展

肺水肿是急性肺损伤(ALI)病理进程中的中心环节,与肺泡毛细血管通透性增加和肺泡内液体清除减少有关。以往对肺泡毛细血管通透性导致的肺水肿研究较多,而对肺泡内液体清除在肺水肿形成中的作用关注不足。肺泡内液体清除受水通道蛋白、钠离子通道、钠钾ATP酶的影响。认识肺泡内液体清除机制有望能为临床医师治疗ALI提供参考。     

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase alpha(1) subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (rho(0) cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase alpha(1) subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-zeta antagonist peptide or with the Na,K-ATPase alpha(1) subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-zeta-mediated phosphorylation of the Na,K-ATPase alpha(1) subunit.     

Constitutive presentation of a natural tissue autoantigen exclusively by dendritic cells in the draining lymph node

The major histocompatibility complex (MHC)-dependent presentation of processed tissue-specific self-antigens can contribute to either peripheral (extrathymic) tolerance or the differentiation of autoreactive T cells. Here, we have studied the MHC class II molecule presentation of gastric parietal cell (PC)-specific H(+)/K(+)-ATPase, which induces a destructive autoimmune gastritis in BALB/c mice lacking CD4(+) CD25(+) regulatory T cells. Immunofluorescence microscopy showed physical association of CD11c(+) dendritic cells (DCs) with PCs in the gastric mucosa. H(+)/K(+)-ATPase protein was found within vesicular compartments of a few CD11c(+) DCs only in the draining gastric lymph node (LN) and these antigen-containing DCs increased markedly in number with the onset of tissue destruction in autoimmune animals. Both CD8alpha(hi) and CD8alpha(lo) gastric DCs, but not peripheral or mesenteric DCs, showed evidence of constitutive in vivo processing and presentation of H(+)/K(+)-ATPase. These data provide direct support for a widely held model of local tissue antigen uptake and trafficking by DCs in normal animals and demonstrate that DCs in the draining LN can present a tissue-specific self-antigen under noninflammatory conditions without fully deleting autoreactive T cells or inducing active autoimmunity.     

Down-regulation of Na+ pump alpha 2 isoform in isoprenaline-induced cardiac hypertrophy in rat: evidence for increased receptor binding affinity but reduced inotropic potency of digoxin

Cardiac hypertrophy in rats induces a down-regulation of Na(+),K(+)-ATPase alpha(2) isoform, although its functional consequences are poorly understood. Using a mathematical modeling approach that allows differentiation between effects elicited at the receptor and postreceptor level, we studied uptake, receptor binding kinetics, and positive inotropism of digoxin in single-pass Langendorff-perfused hearts of vehicle- and isoprenaline-pretreated rats (2.4 mg/kg per day over 4 days). Digoxin outflow concentration and left ventricular developed pressure data were measured for three consecutive doses (15, 30, and 45 microg) in the absence and presence of the reverse mode Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxyl-)phenyl]ethyl isothiourea methansulfonate] (KB-R7943) (0.1 microM) in perfusate. In hypertrophied hearts, 1) the amount of alpha(2) receptors was reduced to 52% of control levels; 2) the digoxin binding affinity was increased 12-fold due to a decrease in dissociation rate constants of alpha(1) and alpha(2) receptors, and 3) inotropic responsiveness to digoxin the was attenuated on the stimulus-response level, where the coupling ratio of stimulus to response was reduced to 38% of control values. Only in the lowest dose level (15 microg) was this decrease in inotropic potency counterbalanced by the increase in receptor affinity. The Na(+),K(+)-ATPase isoform shift was not responsible for the diminished inotropic effect of digoxin. Coadministration of KB-R7943 significantly reduced cellular response generation at higher digoxin doses to the same limiting stimulus-response relationship in both the vehicle and isoprenaline group.     

Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection

Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis–inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury.