Basolateral Cl- transport is stimulated by terbutaline in adult rat alveolar epithelial cells.

Stimulation of adult rat alveolar epithelial cells with terbutaline was previously shown to activate Cl- channels in the apical membrane. In this study, we show that terbutaline stimulates net transepithelial (apical-to-basolateral) Cl- absorption from 0.19 +/- 0.13 to 1.43 +/- 0.31 mmol x cm-2 x hr-1. Terbutaline also increases net Cl- efflux across the basolateral membrane under conditions where an outward [K+] gradient exists and the membrane voltage is clamped at zero mV. When the [K+] gradient is eliminated, the effect of terbutaline on net Cl- efflux is inhibited to the extent that no significant Cl- efflux can be detected across the basolateral membrane. RT-PCR experiments detected mRNA for three KCl cotransport isoforms (KCC1, KCC3 and KCC4) in monolayer cultures of alveolar epithelial cells. Western blot analysis using antibodies to the four cloned isoforms of KCl cotransporters revealed the presence of KCC1 and KCC4 isoforms in monolayer cultures of these cells. These results provide evidence suggesting a role for KCl cotransport in terbutaline-stimulated transepithelial Cl- absorption.

Invited review: lung edema clearance: role of Na(+)-K(+)-ATPase.

Acute hypoxemic respiratory failure is a consequence of edema accumulation due to elevation of pulmonary capillary pressures and/or increases in permeability of the alveolocapillary barrier. It has been recognized that lung edema clearance is distinct from edema accumulation and is largely effected by active Na(+) transport out of the alveoli rather than reversal of the Starling forces, which control liquid flux from the pulmonary circulation into the alveolus. The alveolar epithelial Na(+)-K(+)-ATPase has an important role in regulating cell integrity and homeostasis. In the last 15 yr, Na(+)-K(+)-ATPase has been localized to the alveolar epithelium and its contribution to lung edema clearance has been appreciated. The importance of the alveolar epithelial Na(+)-K(+)-ATPase function is reflected in the changes in the lung's ability to clear edema when the Na(+)-K(+)-ATPase is inhibited or increased. An important focus of the ongoing research is the study of the mechanisms of Na(+)-K(+)-ATPase regulation in the alveolar epithelium during lung injury and how to accelerate lung edema clearance by modulating Na(+)-K(+)-ATPase activity.

Adrenergic regulation of ion transport across adult alveolar epithelial cells: effects on Cl- channel activation and transport function in cultures with an apical air interface.

The effect of beta-adrenergic receptor stimulation on Cl- channel activation was investigated in alveolar epithelial cells grown in monolayer culture and in freshly isolated cells. Monolayers cultured under apical air interface conditions exhibited enhanced amiloride-sensitive Na+ transport compared to apical liquid interface monolayers. Amiloride or benzamil inhibited most (66%) of the basal short circuit current (Isc) with half-maximal inhibitory concentration (IC50) values of 0.62 microm and 0.09 microm respectively. Basolateral addition of terbutaline (2 microm) produced a rapid decrease in Isc followed by a slow recovery that exceeded the basal Isc. When Cl- was replaced with methanesulfonate in either intact monolayers or basolateral membrane permeabilized monolayers, the response to terbutaline (2 microm) was completely inhibited. No effect of terbutaline on amiloride-sensitive Na+ current was detected. beta-Adrenergic agonists and 8-chlorothiophenyl cyclic adenosine monophosphate (8-ctp cAMP) directly stimulated a Cl- channel in freshly isolated alveolar epithelial cells. The current was blocked by glibenclamide (100 microm) and had a reversal potential of -22 mV. No increase in amiloride-sensitve current was detected in response to terbutaline or 8-cpt cAMP stimulation. These data support the conclusion that beta-adrenergic agonists produce acute activation of apical Cl- channels and that monolayers maintained under apical air interface conditions exhibit increased Na+ absorption.

Human surfactant protein a suppresses T cell-dependent inflammation and attenuates the manifestations of idiopathic pneumonia syndrome in mice.

We have previously shown an association between growth factor-induced upregulation of surfactant protein (SP)-A and suppression of alveolar inflammation in our murine model of donor T cell-dependent lung dysfunction after bone-marrow transplantation, referred to as idiopathic pneumonia syndrome (IPS). We hypothesized that SP-A protects the lung in vivo from IPS injury by downregulation of alveolar inflammation. Human SP-A (100 microg), purified by n-butanol extraction or preparative isoelectric focusing, was transtracheally instilled on Day 4 after BMT during a time of in vivo donor T-cell activation. At 48 h after treatment, immunohistochemical staining of lung sections showed that SP-A did not alter T cell- dependent cellular infiltration. However, macrophages from SP-A-instilled mice were less injured and spontaneously produced less tumor necrosis factor-alpha than did cells from buffer-instilled mice. Although exogenous SP-A did not significantly alter bronchoalveolar lavage fluid (BALF) high levels of total protein (TP), an inverse correlation between BALF SP-A and TP concentrations (r = -0.65; P = 0.02) was observed in SP-A-treated but not in buffer-instilled mice. The only difference between the effects of the two sources of SP-A was that butanol-extracted SP-A, but not isoelectric focusing-purified SP-A, suppressed the interferon-gamma/nitric oxide pathway. We conclude that SP-A downregulates T cell-dependent alveolar inflammation by multiple pathways leading to decreased IPS injury.

Cyclophosphamide prevents systemic keratinocyte growth factor-induced up-regulation of surfactant protein A after allogeneic transplant in mice.

We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.

Mechanisms of repair and remodeling following acute lung injury.

At present, we largely lack the ability to correlate the clinical course of ARDS patients with potential factors involved in the biochemical and cellular basis of lung repair. This requires very large patient databases with measurement of many biochemical parameters. Important mechanistic determinants during the repair phase can be sought by correlation with late outcomes, but a large-scale cooperative effort among multiple centers with sharing of follow-up data and patient specimens is essential. We also lack detailed human histologic material from many phases of ARDS and, particularly, know little of the long-term morphologic impact of ARDS in survivors. Establishment of a national registry that follows ARDS survivors and that would seek their cooperation in advance in obtaining autopsy specimens when they die of other causes would be very valuable. Correlating the pathology with their pulmonary function during recovery would give important insights into the reasons for the different patterns of abnormal pulmonary functions. The factors that determine the success of repair are of critical importance in testing new ARDS treatment strategies. Would accelerating the resolution of alveolar edema alter the course of subsequent fibrosis and inflammation? Does surfactant replacement therapy--a costly proposition in adults with ARDS--lead to better long-term outcomes in survivors? How much should we worry about the use of high levels of oxygen for support of arterial partial pressure of oxygen? Is it better to accept hyperoxia to avoid pressure or volume trauma induced by mechanical ventilation with higher minute ventilations? These major management issues all may affect the success of the late repair and recovery process. Intervention trials need to examine the long-term physiologic and functional outcomes.

Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells.

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC(50) values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC(50) values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC(50) of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B(0,+). We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.

Up-regulation of Na,K-ATPase beta 1 transcription by hyperoxia is mediated by SP1/SP3 binding.

The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,K-ATPase beta(1) gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase beta(1) transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5'-deletions of the rat Na,K-ATPase beta(1) promoter-luciferase fusion gene demonstrated promoter activation by hyperoxia between -102 and +151. The hyperoxia response was localized to a 7-base pair region between -62 and -55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at -55 had higher affinity for SP proteins.

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT.

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.

Cl-channel activation is necessary for stimulation of Na transport in adult alveolar epithelial cells.

In this review, we discuss evidence that supports the hypothesis that adrenergic stimulation of transepithelial Na absorption across the alveolar epithelium occurs indirectly by activation of apical Cl channels, resulting in hyperpolarization and an increased driving force for Na uptake through amiloride-sensitive Na channels. This hypothesis differs from the prevailing idea that adrenergic-receptor activation increases the open probability of Na channels, leading to an increase in apical membrane Na permeability and an increase in Na and fluid uptake from the alveolar space. We review results from cultured alveolar epithelial cell monolayer experiments that show increases in apical membrane Cl conductance in the absence of any change in Na conductance after stimulation by selective beta-adrenergic-receptor agonists. We also discuss possible reasons for differences in Na-channel regulation in cells grown in monolayer culture compared with that in dissociated alveolar epithelial cells. Finally, we describe some preliminary in vivo data that suggest a role for Cl-channel activation in the process of amiloride-sensitive alveolar fluid absorption.

Interactions of keratinocyte growth factor with a nitrating species after marrow transplantation in mice.

We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and nitric oxide (. NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-alpha, IFN-gamma, and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible. NO synthase mRNA. KGF also suppressed. NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-alpha, IFN-gamma, and. NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.

Na(+)-K(+)-ATPase gene regulation by glucocorticoids in a fetal lung epithelial cell line.

The Na(+) pump, Na(+)-K(+)-ATPase, along with the Na(+) channel is essential for the removal of alveolar solute and fluid perinatally. Because Na(+)-pump mRNA and activity increase before birth and maternal glucocorticoids (GCs) influence Na(+)-K(+)-ATPase mRNA expression in fetal rat lung, we hypothesized that GCs increased Na(+)-K(+)-ATPase gene expression in a fetal lung epithelial cell line. After 24 h of exposure, dexamethasone increased the steady-state levels of Na(+)-K(+)-ATPase alpha(1) and beta(1) mRNA in a fetal rat lung epithelial cell line in a dose-dependent fashion (10(-7) to 10(-5) M). The maximal increase in mRNA levels was 3. 8-fold for alpha(1) and 2.8-fold for beta(1). The increase in mRNA was detected as early as 6 h for the beta(1)-subunit and 18 h for the alpha(1)-subunit, and both peaked at 24 h. This gene upregulation was not due to increased mRNA stability based on mRNA half-life determination after actinomycin D inhibition. Transfection experiments with alpha(1) and beta(1) promoter-reporter constructs demonstrated 3.2 +/- 0.5- and 2.6 +/- 0.4-fold increases, respectively, in promoter activity, consistent with transcriptional activation of the promoter-reporter construct. These findings, increased promoter activity with no change in stability, indicate that GCs increased Na(+)-K(+)-ATPase transcription in a fetal lung epithelial cell line.

High levels of peroxynitrite are generated in the lungs of irradiated mice given cyclophosphamide and allogeneic T cells. A potential mechanism of injury after marrow transplantation.

In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (.NO) production, and by Cy, which stimulates superoxide production.NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells +/- Cy. AMs obtained on Day 7 after BMT from mice receiving T cells +/- Cy spontaneously generated between 20 and 40 microM nitrite in culture, versus < 2 microM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived.NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels.

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current (Isc; 60%) was inhibited by amiloride (IC50 0. 96 microM) or benzamil (IC50 0.5 microM). Basolateral addition of terbutaline (2 microM) produced a rapid decrease in Isc, followed by a slow recovery back to its initial amplitude. When Cl- was replaced with methanesulfonic acid, the basal Isc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of -28 mV. Increasing Cl- concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl- conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that beta-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl- permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.

Oxidant effects on epithelial Na,K-ATPase gene expression and promoter function.

The lung epithelium resorbs alveolar fluid through combined action of sodium channels and the sodium pump, Na,K-ATPase. The lung often is exposed to hyperoxia in disease states and hyperoxia generates a mixture of reactive oxygen species. In vivo and in vitro exposure of rat lung and alveolar type II cells, respectively, increases gene expression of both alpha-1 and beta-1 subunits of the sodium pump. In contrast to the primary type II cells, several type II cell lines did not increase sodium pump gene expression with hyperoxia, but the renal tubular epithelial MDCK cell line did. Using promoter-receptor constructs transfected into MDCK cells, hyperoxia did not markedly increase transcription of the alpha-1 subunit but doubled transcription of the beta-1 subunit gene. Using 5'-deletion constructs, the region required for the beta-1 increase was localized to a 40-base pair region from -44/-84. The hyperoxic responsiveness of this region was confirmed using constructs with one or two copies of this region placed in minimal promoter-luciferase reporters. This 5' promoter region contains a consensus binding sequence for SP-1, a basal transcription factor but not for binding of other known transcription factors. Thus, hyperoxia induces Na,K-ATPase beta-1 promoter transcription, likely acting through a novel mechanism.

The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells.

Previously it was reported that hyperoxia induced death of the human lung adenocarcinoma cell line (A549 cells) by necrosis, not by apoptosis. This study examined proliferation and death of untransformed human small airway epithelial (SAE) cells in normoxia or hyperoxia in comparison with A549 cells. We tested the hypothesis that SAE cells respond differently to hyperoxic injury than do A549 cells. We measured total cell number and viability, thymidine incorporation (SAE cells only), lactate dehydrogenase (LDH) release, and apoptotic changes as markers for cell proliferation and death. Protective effects of antioxidant vitamins also were examined in SAE cells. In normoxia, subconfluent SAE cells had less apoptosis and fewer detached cells, but higher thymidine incorporation than did near-confluent cells. Hyperoxia suppressed thymidine incorporation and augmented apoptosis in both subconfluent and near-confluent SAE cells. Hyperoxia decreased the total cell number only in subconfluence, whereas SAE cell viability declined with hyperoxia in near confluence, but not in subconfluence. For SAE cells, necrosis assessed by LDH release was minimal in all conditions and was not augmented by hyperoxia in SAE cells. In contrast, normoxic A549 cells proliferated more rapidly than did SAE cells with a large number of cells detached during the culture. A549 cells underwent necrotic cell death under confluent or in hyperoxic conditions, but had much less apoptotic cell death. In SAE cells, vitamin E partially prevented the decline of thymidine incorporation with hyperoxia in subconfluence and protected against apoptotic changes with hyperoxia in both subconfluent and near-confluent conditions. Vitamin C prevented apoptosis with hyperoxia only in near-confluent SAE cells. Thus, SAE cells maintained balanced apoptosis and cell proliferation that were altered by cell density and hyperoxia and demonstrated very little necrosis with hyperoxia. Although A549 cells underwent cell death mainly by necrosis, they also were influenced by cell density and hyperoxia. Cell density also determined specific antioxidant vitamin protection in SAE cells.