Interdependency of -Adrenergic Receptors and CFTR in Regulation of Alveolar Active Na+ Transport
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循环研究杂志 2005年第5期
The Pulmonary and Critical Care Medicine (G.M.M., Y.A., L.W., J.I.S.), Northwestern University Feinberg School of Medicine, Chicago, Ill
Evanston Northwestern Healthcare Research Institute (M.J., C.K., R.B.), Evanston Ill
Pulmonary, Allergy
Critical Care Medicine (A.T.A., F.J.M., E.R.L., P.F.), Columbia University Medical Center, New York, NY
University of Illinois College of Medicine (V.D.), Chicago, Ill
the University of Iowa College of Medicine (J.Z.,G.T.), Iowa City, Iowa.
Abstract
-Adrenergic receptors (AR) regulate active Na+ transport in the alveolar epithelium and accelerate clearance of excess airspace fluid. Accumulating data indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) is important for upregulation of the active ion transport that is needed to maintain alveolar fluid homeostasis during pulmonary edema. We hypothesized that AR regulation of alveolar active transport may be mediated via a CFTR dependent pathway. To test this hypothesis we used a recombinant adenovirus that expresses a human CFTR cDNA (adCFTR) to increase CFTR function in the alveolar epithelium of normal rats and mice. Alveolar fluid clearance (AFC), an index of alveolar active Na+ transport, was 92% greater in CFTR overexpressing lungs than controls. Addition of the CleC channel blockers NPPB, glibenclamide, or bumetanide and experiments using CleC free alveolar instillate solutions indicate that the accelerated AFC in this model is due to increased CleC channel function. Conversely, CFTR overexpression in mice with no 1- or 2-adrenergic receptors had no effect on AFC. Overexpression of a human 2AR in the alveolar epithelium significantly increased AFC in normal mice but had no effect in mice with a non-functional human CFTR gene (508 mutation). These studies indicate that upregulation of alveolar CFTR function speeds clearance of excess fluid from the airspace and that CFTRs effect on active Na+ transport requires the AR. These studies reveal a previously undetected interdependency between CFTR and AR that is essential for upregulation of active Na+ transport and fluid clearance in the alveolus.
Key Words: pulmonary edema cystic fibrosis transmembrane conductance regulator alveolar fluid clearance chloride channel -adrenergic receptors
Introduction
-Adrenergic receptors (AR) on alveolar epithelial cells upregulate active Na+ transport and accelerate clearance of excess fluid from the alveolar airspace. Data from in vitro and in vivo models indicate that AR signaling increases epithelial Na+ channel number and open probability and promotes transcription and trafficking of Na,K-ATPases to the cell membrane of alveolar epithelial cells.1eC3 Maintenance of electroneutrality across the alveolar epithelium requires that CleC exit the airspace via trans- and paracellular pathways.4 Accumulating data from in vitro and in vivo studies indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is present in alveolar epithelial cells and provides a path for CleC transport to clear excess alveolar fluid, although it may not be needed for maintenance of alveolar fluid balance in the normal/uninjured lung.5
We have recently reported that the 2AR is essential for upregulation of alveolar active Na+ transport.6 In these studies, we observed that 2AR signaling is required to maintain normal expression or function of amiloride-sensitive Na+ transport and basolateral Na,K-ATPase function in the distal mouse lungs. Data from both airway and alveolar epithelial cells indicate that AR signaling increases CleC flux through CFTR, which is present at low levels in the apical cell membrane of human and rodent alveolar epithelial cells.7,8 Single cell and monolayer studies of rat and rabbit alveolar epithelial cells indicate that these cells can reabsorb CleC through a cAMP-sensitive pathway.8,9 This body of data led us to hypothesize that CFTR may play a role in 2AR-mediated effects on alveolar active Na+ transport.
In the present study, we tested the effects of upregulation of CFTR function on alveolar active Na+ transport in normal rats and mice, and mice with targeted deletions of the AR or with a nonfunctional human CFTR gene (CFTR 508). These studies reveal an interdependency between CFTR and the 2AR where both proteins are required to affect increases in AR-driven alveolar active Na+ transport.
Materials and Methods
Adenovirus Propagation and Purification
Replication-incompetent E1aeC/E3eC human type 5 adenoviruses containing either a full-length human CFTR cDNA under the control of a human CMV immediate-early promoter enhancer gene (adCFTR, from Drs M. Welsh and J. Zabner, University of Iowa, Iowa City, Iowa), a human 2AR cDNA under the control of a human CMV immediate-early promoter enhancer gene (ad2AR, from Drs R. Lefkowitz and W. Koch, Duke University, Durham, NC), or no cDNA (adNull) were propagated and purified as previously described.10,11 All viruses used in this study were from single preparations and were free of signs of replication-competent adenovirus to a maximum dilution of 109.
Animals
The use of animals for this study was approved by the Evanston Northwestern Healthcare (ENH), Northwestern University, and Columbia University Institutional Animal Use and Care Committees. Mice homozygous for deletions of both the 1- and 2-adrenergic receptors (1AReC/eC/2AReC/eC) and strain-specific 1AR+/+/2AR+/+ control mice (C57Bl6/J-DBA/2-129SvJ) were provided by Dr B. Kobulka (Stanford University, Stanford, Calif).12 Transgenic mice homozygous for the 508 mutation in the CFTR gene (CFTReC/eC) and wild-type (CFTR+/+) littermate controls were from Dr J. Engelhardt (University of Iowa, Iowa City, Iowa).13 Concurrently maintained sentinel animals were without signs of specific viral or bacterial pathogens. C57bl6 mice were from Harlan Laboratories, Indianapolis, Ind. All mice used in these studies were 8- to 12-week-old males weighing 20 to 25 g. Rats used in this study were adult, male Sprague-Dawley rats (Harlan Laboratories) weighing 275 to 300 g.
Adenovirus Delivery to Rat and Mouse Lungs
Adenovirus was delivered to rats and mice using previously described methods.6,10,14 Briefly, these methods use a surfactant-based vehicle that produces transgene expression that is confined to the alveolar epithelium. Rats received 5x1010 viral particles and mice 1x1010 viral particles. In all experiments, mice and rats infected with ad2AR or adCFTR were compared with rats treated with vehicle alone (sham) or infected with a first generation adenovirus that expresses no transgene (adNull). This strategy and inclusion of 7 days of after infection recovery has proven effective in controlling for the effects of the host response to these adenovectors in prior studies.6,10,15eC17 We have previously shown that sham infection has no measurable effect on alveolar fluid clearance (AFC) in rats and mice; thus, uninfected controls were not used in this study.6,10,15eC18
Measurement of AFC in Isolated Rat Lungs
The isolated lung preparation used in this study has been previously used to measure alveolar active Na+ transport in rats infected with adenoviruses.10,15eC17,19eC21 Briefly, change in concentration of a protein marker (Evan’s blue tagged albumin) instilled into the airspace compartment of isolated and perfused rats lungs is used to calculate change in alveolar volume over a 60-minute experimental period. Fluorescene isothiocyanate (FITC)eCtagged albumin was added to the perfusate to monitor leakage of protein from the vascular compartment into the airspace to produce an index of alveolar permeability.
Rat Treatment Protocols
AFC was measured in sham, adNull, and adCFTR-infected rats (9 animals/group). An additional 5 rats per group were studied with amiloride (10eC6 mol/L) in the alveolar instillate during clearance measurements. To estimate CleC channel function in sham, adNull, and adCFTR-infected rat lungs the CleC transport inhibitors 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 10eC4 mol/L), glibenclamide (10eC4 mol/L), or bumetanide (10eC4 mol/L) were individually added to the alveolar instillate during clearance measurements. The Na+ channel blocker amiloride (10eC6 mol/L) was included in the alveolar instillate in some experiments.
AFC Measurements in Live Mice
The method of measurement used in this study was modified from Hardiman et al and has been described elsewhere.6,22,23 This method is similar to that used in rats except that 300 e蘈 of Evan’s blue-tagged albumin is instilled into lungs of sedated, mechanically ventilated mice that are maintained supine, and clearance is measured over a 30-minute period. Assessment of CleC channel function in mice was via use of a CleC-free instillate solution where NaCl was replaced with equimolar amounts of NaGluconate without altering osmolarity. In some mice, forskolin (10eC4 mol/L) was included in the instillate solution during clearance measurements. Experiments using CleC-free instillate were performed with a positive end-expiratory pressure of 3 cmH2O above ambient pressure using a water valve attached to the expiratory port of a rodent ventilator.
Reverse Transcriptase PCR
Total RNA from peripheral (distal 2 to 3 mm) and proximal (central airways) lung tissue and liver was isolated using RNA-zol B (Tel-Test, Inc). RNA was treated with DNAse I (50 U, Sigma,), twice extracted with phenol-chloroform, precipitated, and resuspended in 20 e蘈 of RNAse-free water before first strand cDNA synthesis using oligo-dT12-16. PCR was performed using 2 e蘈 of the reaction mixture, 30 cycles of amplification, and an annealing temperature of 58°C. Primer sequences used to detect human CFTR mRNA were as follows: upstream 5'ACGCTTCAGGCACGAA and downstream 5'CGGCTACTCCCACGTAA.
Cell Membrane Isolation
Whole-cell membrane proteins and membrane fractions enriched for the basolateral membrane domain were produced from tissue collected from the distal 2 to 3 mm of right rat lungs after serial bronchoalveolar lavage and perfusion of the pulmonary artery with PBS, as previously described.10,16,17,24
Western Analysis
Twenty micrograms of whole-cell membrane protein was separated with 4% to 12% SDS-PAGE (Invitrogen Life Technology), electrophoretically transferred to nitrocellulose and probed with a mouse monoclonal antieChuman CFTR antibody (R&D Systems), a rabbit antieCrat ENaC antibody (Affinity Bioreagents), a rabbit antieCNa,K-ATPase 1 antibody (K. Geering, University of Lausanne, Switzerland)25 or a mouse monoclonal anti-actin antibody (Chemicon International). Protein bands were visualized using peroxidase coupled secondary antibodies and a chemiluminescence detection kit (Pierce).
Na,K-ATPase Function (Pi Liberation From ATP) in the Distal Lung
Twenty micrograms of basolateral cell membrane protein isolated from the peripheral lung (distal 2 to 3 mm) of rats was resuspended in 100 e蘈 of a high [Na+]/low [K+]/high [ATP] reaction buffer (in mmol/L: 50 Tris-HCl pH 7.4, 50 NaCl, 5 KCl, 10 Mg2Cl, 1 EGTA, 10 Na2ATP), with [-32P]-ATP (3.3 e藽i/mL) as previously described.16,17 These conditions maximize Na,K-ATPase activity (Vmax) and produce an index of functional, membrane-bound receptor number. Triplicate samples were placed at eC20°C for 15 minutes before incubation for 15 minutes at 37°C. The reaction was terminated by the addition of 5% TCA/10% charcoal and cooling to 4°C. The charcoal phase containing unhydrolyzed nucleotide was separated by centrifugation (12 000g for 5 minutes) and the liberated 32P quantified. Na,K-ATPase activity was calculated as the difference between the test samples (total/nonspecific ATPase activity) and samples assayed in reaction buffer with 2.5 mmol/L ouabain but devoid of Na+ and K+ (non-Na,K-ATPase ATPase activity). Results are expressed as mean nmol of Pi/mg of protein per hour of triplicate measurements from 3 rats per group.
Immunohistochemistry for CFTR
Mouse lungs (C57b6) were fixed in 2% formaldehyde/0.2% glutaraldehyde imbedded in paraffin and sectioned (5 e). Deparafinized and rehydrated sections were blocked at room temperature for 1 hour with 5% normal goat serum in Superblock before incubation with anti-CFTR antibodies (13-1 and 24-1, R&D Systems, 1:100 dilution; MM13eC4 and M3A7, Upstate USA, Inc, 1:200 dilution) at 37°C for 2 hours and then washed 5 times with PBS. Immunodetection was with Alexa568 (Molecular Probe, 1:1000) for 1 hour (at 37°C) followed by washing 5 times with PBS. The slides were then mounted with Vectashield (Vector Labs, Inc).
Data Analysis
All values are reported as mean±standard deviation (SD). Statistical significance was defined as P<0.05.
Results
Transgene Expression
To produce a model of alveolar CFTR overexpression, a first generation adenovirus that expresses a human CFTR cDNA under the control of a human CMV promoter-enhancer element (adCFTR) was used to transduce the lungs of normal rats (Figure 1). This vector has been shown to increase CFTR function in airway epithelial cells, animal models, and humans with cystic fibrosis.26eC29 Human CFTR mRNA was detected in both proximal and distal rat lung tissue 7 days after infection with adCFTR using rtPCR and human CFTR-specific primers (Figure 1A). No human CFTR mRNA was observed in livers of adCFTR-infected rats or in lungs and livers from adNull-infected controls. Increased CFTR protein expression was noted in cell membranes isolated from the distal lungs of adCFTR-infected rats (Figure 1B). Immunostaining of adCFTR-infected C57b6 mouse lungs using antieChuman CFTR primary antibodies demonstrated patchy immunoreactivity in a linear pattern that surrounded the airspace, consistent with transduction of all alveolar epithelial cells (Figure 1C).
Effects of CFTR Overexpression on AFC in Rats
AFC in rats infected with adCFTR (5x101 viral particles) was 92% greater (0.98±0.11 mL/hour) than adNull (0.51±0.06 mL/hour) and sham-infected controls (0.51±0.02, P<0.002 adCFTR versus sham and adNull) (Figure 2). No flux of albumin from the pulmonary vasculature into the airspace was measurable in any of the experimental groups implying that alveolar barrier function was not altered by gene transfer or CFTR overexpression. To test if CFTR gene transfer affects alveolar CleC channel function, we conducted experiments using 3 established CleC channel inhibitors in the alveolar instillate during clearance measurements. NPPB did not affect AFC in sham- and adNull-infected lungs, whereas glibenclamide and bumetanide reduced it by 20% (Figure 3A through 3C). Glibenclamide, bumetanide, and NPPB reduced AFC in adCFTR-infected lungs by 55% to 60%. Net clearance in adCFTR-infected lungs treated with these agents was the same as similarly treated sham- and adNull-infected controls; thus, CleC channel blockade completely negated the effects of CFTR overexpression.
To further evaluate if CFTR gene transfer alters epithelial CleC channel function, AFC was measured using an instillate solution free of CleC (Figure 3D). Doing so resulted in CleC flux along its concentration gradient from the interstitial into the alveolar airspace and has been shown to cause fluid accumulation in the airspace.4 These experiments were conducted using C57bl6 mice and a positive end-expiratory pressure of 3 cmH2O, which increased basal AFC to 30.29±4.5%/30 minutes. This was substantially greater than that reported previously by our group6 but is consistent with recent data from Hardiman.23 The absence of CleC caused airspace fluid accumulation (AFC: eC8.8±3.7/%30 minutes) that doubled to eC17.5±2.8%/30 minutes when the adenylyl cyclase activator forskolin was included in the instillate of adNull-infected mice. Basal and forskolin-stimulated AFC in adCFTR mice was significantly more negative than adNull (eC19.1±2.7%/30 minutes and eC22.7±5.2%/30 minutes, respectively). These findings are consistent with increased function of a cAMP sensitive CleC channel.
Effects of CFTR Overexpression on Na+ Channel and Na,K-ATPase in Rats
Amiloride (10eC6 mol/L) was included in the alveolar instillate during clearance measurements to assess amiloride-sensitive Na+ channel function. As can be seen in Figure 4A, amiloride reduced AFC to a greater degree in adCFTR-infected lungs than in sham- and adNull-infected controls (AFC: adCFTR, eC57% to 0.42±0.11 mL/hour; adNull, eC46% to 0.26±0.03 mL/hour; sham, eC49% to 0.27±0.07 mL/hr) (P<0.05 adCFTR versus adNull or sham) (Figure 4A). However, the absolute clearance rate in amiloride-treated adCFTR-infected lungs (0.42±0.11 mL/hour) remained greater than similarly treated sham (0.27±0.07 mL/hour) and adNull controls (0.26±0.03 mL/hour) (P<0.03 adCFTR versus sham or adNull). Western analysis for Epithelial Na+ Channel subunit (ENaC) using whole-cell membranes from the peripheral lung did not reveal significant differences in expression between adNull and adCFTR infected lungs (Figure 4B). Thus, CFTR gene transfer increases both amiloride-sensitive and insensitive Na+ channel function in rats. Whether this is due to changes in ENaC expression, the biophysical properties/amiloride sensitivity of ENaC, or upregulation of an amiloride-insensitive apical Na+ entry pathway is not yet known.
The effect of CFTR gene transfer on Na,K-ATPase activity was assessed by measuring ouabain-sensitive liberation of inorganic phosphate (Pi) from ATP in the presence of substrate independent conditions (high [Na+]/low [K+]/high [ATP]) that allow the Na+ pump to function maximally (Vmax). Under these conditions, the determinate of Pi release is the number of functional Na,K-ATPases in basolateral membranes from the peripheral lung. AdCFTR-infected rat lungs had up to 66% more functional Na,K-ATPases in basolateral membranes than sham- and adNull-infected controls (2.91±0.32, 1.57±0.23, and 1.75±0.42 nmol Pi/mg protein per hour, respectively; P<0.02 adCFTR versus sham and adNull) (Figure 5A). Western analysis likewise showed increased expression of the Na,K-ATPase 1 subunit in adCFTR-infected lungs (Figure 5B). These findings indicate that upregulation of CleC channel function results in parallel changes of other transport proteins.
CFTR Gene Transfer to Alveoli of Mice With Targeted Deletions of the 1- and 2-Adrenergic Receptors
We have recently reported that mice with targeted deletions of the 1 and 2 adrenergic receptors (1AReC/eC/2AReC/eC) have decreased basal AFC, Na,K-ATPase function, and amiloride sensitivity.6 Rescue of alveolar epithelial 2AR function normalizes these processes confirming that these mice retain G-protein signaling capability and can upregulate lung ion transport proteins. To test if changes in active transport after CFTR gene transfer require 2AR function, we infected mice with 1x1010 viral particles of adCFTR 7 days before study using a recently described surfactant-based delivery system.6 Fluid clearance in this model was measured using a "live" mechanically ventilated intact lung model that preserves oxygenation, ventilation, and serum pH.22,23 CFTR gene transfer increased AFC in strain-specific 1AR+/+/2AR+/+ mice by 36% (from 22.2±3.0% to 30.3±2.9%; P<0.02 versus uninfected, sham, and adNull infected 1AR+/+/2AR+/+’; Figure 6A). This increase was less than that noted in rats (Figure 2) and is likely due to lower transduction efficiency (70%6 versus >90%10,14) and perhaps decreased dependence on CFTR for AFC in mice than rats. Importantly, CFTR gene transfer to the alveolar epithelium of 1AReC/eC/2AReC/eC mice had no effect on AFC measured 7 days after gene transfer. AdNull infection did not affect AFC at this postinfection time-point in either the 1AR+/+/2AR+/+ or 1AReC/eC/2AReC/eC groups. Clearance in these groups was the same as untreated/uninfected mice in a prior study.2
2AR Gene Transfer to the Alveoli of Mice With a Nonfunctional Human CFTR
To further approach the question of a functional interaction between 2AR and CFTR, we transduced the alveolar epithelium of mice that have no mouse CFTR gene (CFTReC/eC) and are homozygous for a nonfunctional human CFTR gene (508 mutation) with an adenovirus that expresses a human 2AR gene (ad2AR) (Figure 6B). We have shown that this approach increases 2AR expression only in the alveolar epithelium and upregulates 2AR function and alveolar active Na+ transport in both wild-type and 1AReC/eC/2AReC/eC mice.6 Increased alveolar -receptor function in this model appears to be due to increased numbers of 2ARs in the cell membrane and enhanced sensitivity to endogenous catecholamines. As can be seen in Figure 6B, 2AR gene transfer did not affect AFC in CFTReC/eC mice but increased it by 42% in wild-type controls (from 21.7±4.2% to 30.7±5.6%; P=0.04 versus sham-infected CFTR+/+). Clearance in sham- and adNull-nfected CFTReC/eC mice was similar to sham (Figure 6B) and uninfected2 wild-type mice consistent with other reports showing that CFTR may not be required for maintenance of basal alveolar active Na+ transport.30 These data, combined with that from experiments of CFTR gene transfer to 1AReC/eC/2AReC/eC mice suggest that 2AR mediates increases in alveolar active Na+ transport via a CFTR-dependent pathway and that CFTR function requires the presence of the 2AR.
Discussion
In the current study, we tested if upregulation of CFTR function, via adenoviral-mediated overexpression, alters alveolar active Na+ transport in rodents. To affect this goal, we used previously tested adenovectors and established delivery strategies that produce transgene expression in both type 1 and 2 alveolar epithelial cells in rats and mice (Figure 1C).16 Data from other groups indicates that the 2AR is present in both type 1 and 2 epithelial cells,30,31 thus it is likely that the methods used in these experiments result in concomitant expression of CFTR and 2AR in both types of alveolar epithelial cells.
The functional effects of CFTR gene transfer in rats were assessed by measuring the capacity of the lung to clear fluid instilled into the lung using a well-described isolated rat lung model.10 This experimental design is based on the principal that AFC is an index of active, vectorial Na+ transport by alveolar epithelial cells. Using this approach, we observed that overexpression of a human CFTR increases alveolar active Na+ transport by nearly 100% in normal rats (Figure 2) and by 42% in mice (Figure 6B). Inclusion of CleC channel blockers in the alveolar instillate during clearance measurements obviated the effects of CFTR overexpression (Figure 3). None of the blocking agents used is perfectly specific for CFTR, thus additional studies were conducted in mice using a CleC-free instillate. This model results in CleC and fluid flux into the airspace and can be used to assess CleC channel function.4 Using this method, we noted that airspace fluid accumulation was greater in adCFTR-infected mice than in untreated and forskolin-treated adNull controls (Figure 3D). The inclusion of the adenylyl cyclase activator forskolin in the airspace of adCFTR-infected mice produced significant incremental fluid accumulation consistent with increased function of a cAMP-regulated CleC channel such as CFTR.
To probe how CFTR overexpression effects Na+ channel function, we included amiloride in the alveolar compartment during clearance measurements in rats (Figure 4A). Amiloride reduced alveolar active Na+ transport in lungs overexpressing CFTR, implying that increased CFTR function results in increased amiloride-sensitive Na+ channel (ie, epithelial Na+ channel) function (Figure 4A). Absolute clearance rates in amiloride-treated adCFTR lungs were greater than in similarly treated controls, suggesting that CFTR gene transfer also upregulates amiloride-insensitive Na+ entry pathways (Figure 4A). CFTR-infected rats also had significant increases in the number of functional Na,K-ATPases in basolateral cell membranes (Figure 4B). How then does CFTR overexpression increase vectorial ion transport We speculate that overexpression of CFTR increases the number of open/activated CFTRs in the cell membraneeCenhancing CleC entry and results in upregulation of amiloride-sensitive and insensitive Na+ entry pathways. This hypothesis requires that CFTR activate ENaC and other Na+ entry pathways and is in contradistinction to data from MDCK cells.32 Importantly, our data suggests that the interaction of CFTR with apical Na+ channels in alveolar epithelial cells might be markedly different than in proximal airway epithelial cells. Irrespective of this speculation, increased Na+ flux in the setting of CFTR overexpression results in upregulation of basolateral Na,K-ATPase capacity. The net result of these effects is increased vectorial Na+ flux and accelerated AFC.
Both 1- and 2-adrenergic agonists upregulate alveolar active Na+ transport in experimental models16 and humans.33,34 These agonists increase trafficking of Na,K-ATPases to the basolateral cell membrane, cause trafficking to the cell membrane and opening of epithelial Na+ channels, and increase CleC flux through CFTR.2 To probe for a functional interaction between alveolar AR and CFTR, we tested the effects of CFTR overexpression in mice with targeted deletions of both the 1- and 2- adrenergic receptor (1AReC/eC/2AReC/eC). We have previously shown that these mice have normal total lung cAMP levels and retain G-protein signaling and adenylyl cyclase function but have much reduced basal alveolar active Na+ transport rates that can be normalized via rescue of 2AR function into the alveolar epithelium.6 CFTR overexpression in 1AReC/eC/2AReC/eC mice had no effect on alveolar active Na+ transport (Figure 6A). Thus, CFTR overexpression, in and of itself, does not alter vectorial ion flux across the alveolar epithelium. This observation caused us to test the effect of 2AR gene transfer to the alveolar epithelium of 508 mice. These crucial studies revealed that 2AR gene transfer in the absence of functional CFTR does not accelerate alveolar active Na+ transport as it does in wild-type controls. Recently, Fang and colleagues35 reported that CFTReC/eC mice are unable to compensate for increased lung water in a model of hydrostatic pulmonary edema and, importantly, do not accelerate alveolar active Na+ transport in response to -agonist treatment. We believe that the data from the present study and that of Fang support a new paradigm where CFTR and the AR are functionally interdependent and both are required to upregulate alveolar active Na+ transport in the setting of pathological increases of fluid in the alveolar airspace. Presumptive mechanisms for this interaction have been suggested by Naren and colleagues who demonstrated that in airway epithelial cells (calu-3) 2ARs physically associate with CFTR through the PDZ domain of ezrin/radixin/moesin-binding phosphoprotein 50.36 The formation of this macromolecular complex facilitates access of cAMP-activated protein kinase to R domain sites on CFTR and is required for cAMP-mediated regulation of CFTR.
The findings of this study raise the possibility that CleC transport may be a rate-limiting step for vectorial ion transport in the alveolar epithelium. While possible, we believe that alveolar active transport requires several key transport proteins (eg, ENaC, CFTR, Na,K-ATPase) that must interact to generate a transepithelial NaCl gradient, and that impairment of any of these protein families will impede AFC. Likewise, based on data from other studies, upregulation of single member of the transport pathway results in compensatory augmentation of other pathway components.6,10,11,16eC18
The present study provides new evidence that CFTR is an important contributor to the pathway by which the lung clears fluid from the alveolar airspace and that there exists an interdependence between 2AR and CFTR where both are required to upregulate alveolar active Na+ transport. These studies raise concern that the alveolar epithelium of patients with cystic fibrosis may lack the capacity to upregulate alveolar active Na+ transport in response to lung injury. The results of this study also suggest that upregulation of alveolar CFTR function might be an adjunct for improving the lung’s ability to clear pulmonary edema.
Acknowledgments
This work was supported by HL-66211, HL-71042, HL-19094, American Heart Association, and the Evanston Northwestern Healthcare Research Institute. We thank Dr K. Geering (Lausanne University) for providing us with antieCNa,K-ATPase 1 antibody.
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Hardiman KM, McNicholas-Bevensee CM, Fortenberry J, Myles CT, Malik B, Eaton DC, Matalon S. Regulation of amiloride-sensitive Na+ transport by basal nitric oxide. Am J Respir Cell Mol Biol. 2003; 30: 720eC728.
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Naren AP, Cobb B, Li C, Roy K, Nelson D, Heda GD, Liao J, Kirk KL, Sorscher EJ, Hanrahan J, Clancy JP. A macromolecular complex of beta 2 adrenergic receptor, CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA. Proc Natl Acad Sci U S A. 2003; 100: 342eC346.(Gkhan M. Mutlu, Yochai Ad)
Evanston Northwestern Healthcare Research Institute (M.J., C.K., R.B.), Evanston Ill
Pulmonary, Allergy
Critical Care Medicine (A.T.A., F.J.M., E.R.L., P.F.), Columbia University Medical Center, New York, NY
University of Illinois College of Medicine (V.D.), Chicago, Ill
the University of Iowa College of Medicine (J.Z.,G.T.), Iowa City, Iowa.
Abstract
-Adrenergic receptors (AR) regulate active Na+ transport in the alveolar epithelium and accelerate clearance of excess airspace fluid. Accumulating data indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) is important for upregulation of the active ion transport that is needed to maintain alveolar fluid homeostasis during pulmonary edema. We hypothesized that AR regulation of alveolar active transport may be mediated via a CFTR dependent pathway. To test this hypothesis we used a recombinant adenovirus that expresses a human CFTR cDNA (adCFTR) to increase CFTR function in the alveolar epithelium of normal rats and mice. Alveolar fluid clearance (AFC), an index of alveolar active Na+ transport, was 92% greater in CFTR overexpressing lungs than controls. Addition of the CleC channel blockers NPPB, glibenclamide, or bumetanide and experiments using CleC free alveolar instillate solutions indicate that the accelerated AFC in this model is due to increased CleC channel function. Conversely, CFTR overexpression in mice with no 1- or 2-adrenergic receptors had no effect on AFC. Overexpression of a human 2AR in the alveolar epithelium significantly increased AFC in normal mice but had no effect in mice with a non-functional human CFTR gene (508 mutation). These studies indicate that upregulation of alveolar CFTR function speeds clearance of excess fluid from the airspace and that CFTRs effect on active Na+ transport requires the AR. These studies reveal a previously undetected interdependency between CFTR and AR that is essential for upregulation of active Na+ transport and fluid clearance in the alveolus.
Key Words: pulmonary edema cystic fibrosis transmembrane conductance regulator alveolar fluid clearance chloride channel -adrenergic receptors
Introduction
-Adrenergic receptors (AR) on alveolar epithelial cells upregulate active Na+ transport and accelerate clearance of excess fluid from the alveolar airspace. Data from in vitro and in vivo models indicate that AR signaling increases epithelial Na+ channel number and open probability and promotes transcription and trafficking of Na,K-ATPases to the cell membrane of alveolar epithelial cells.1eC3 Maintenance of electroneutrality across the alveolar epithelium requires that CleC exit the airspace via trans- and paracellular pathways.4 Accumulating data from in vitro and in vivo studies indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is present in alveolar epithelial cells and provides a path for CleC transport to clear excess alveolar fluid, although it may not be needed for maintenance of alveolar fluid balance in the normal/uninjured lung.5
We have recently reported that the 2AR is essential for upregulation of alveolar active Na+ transport.6 In these studies, we observed that 2AR signaling is required to maintain normal expression or function of amiloride-sensitive Na+ transport and basolateral Na,K-ATPase function in the distal mouse lungs. Data from both airway and alveolar epithelial cells indicate that AR signaling increases CleC flux through CFTR, which is present at low levels in the apical cell membrane of human and rodent alveolar epithelial cells.7,8 Single cell and monolayer studies of rat and rabbit alveolar epithelial cells indicate that these cells can reabsorb CleC through a cAMP-sensitive pathway.8,9 This body of data led us to hypothesize that CFTR may play a role in 2AR-mediated effects on alveolar active Na+ transport.
In the present study, we tested the effects of upregulation of CFTR function on alveolar active Na+ transport in normal rats and mice, and mice with targeted deletions of the AR or with a nonfunctional human CFTR gene (CFTR 508). These studies reveal an interdependency between CFTR and the 2AR where both proteins are required to affect increases in AR-driven alveolar active Na+ transport.
Materials and Methods
Adenovirus Propagation and Purification
Replication-incompetent E1aeC/E3eC human type 5 adenoviruses containing either a full-length human CFTR cDNA under the control of a human CMV immediate-early promoter enhancer gene (adCFTR, from Drs M. Welsh and J. Zabner, University of Iowa, Iowa City, Iowa), a human 2AR cDNA under the control of a human CMV immediate-early promoter enhancer gene (ad2AR, from Drs R. Lefkowitz and W. Koch, Duke University, Durham, NC), or no cDNA (adNull) were propagated and purified as previously described.10,11 All viruses used in this study were from single preparations and were free of signs of replication-competent adenovirus to a maximum dilution of 109.
Animals
The use of animals for this study was approved by the Evanston Northwestern Healthcare (ENH), Northwestern University, and Columbia University Institutional Animal Use and Care Committees. Mice homozygous for deletions of both the 1- and 2-adrenergic receptors (1AReC/eC/2AReC/eC) and strain-specific 1AR+/+/2AR+/+ control mice (C57Bl6/J-DBA/2-129SvJ) were provided by Dr B. Kobulka (Stanford University, Stanford, Calif).12 Transgenic mice homozygous for the 508 mutation in the CFTR gene (CFTReC/eC) and wild-type (CFTR+/+) littermate controls were from Dr J. Engelhardt (University of Iowa, Iowa City, Iowa).13 Concurrently maintained sentinel animals were without signs of specific viral or bacterial pathogens. C57bl6 mice were from Harlan Laboratories, Indianapolis, Ind. All mice used in these studies were 8- to 12-week-old males weighing 20 to 25 g. Rats used in this study were adult, male Sprague-Dawley rats (Harlan Laboratories) weighing 275 to 300 g.
Adenovirus Delivery to Rat and Mouse Lungs
Adenovirus was delivered to rats and mice using previously described methods.6,10,14 Briefly, these methods use a surfactant-based vehicle that produces transgene expression that is confined to the alveolar epithelium. Rats received 5x1010 viral particles and mice 1x1010 viral particles. In all experiments, mice and rats infected with ad2AR or adCFTR were compared with rats treated with vehicle alone (sham) or infected with a first generation adenovirus that expresses no transgene (adNull). This strategy and inclusion of 7 days of after infection recovery has proven effective in controlling for the effects of the host response to these adenovectors in prior studies.6,10,15eC17 We have previously shown that sham infection has no measurable effect on alveolar fluid clearance (AFC) in rats and mice; thus, uninfected controls were not used in this study.6,10,15eC18
Measurement of AFC in Isolated Rat Lungs
The isolated lung preparation used in this study has been previously used to measure alveolar active Na+ transport in rats infected with adenoviruses.10,15eC17,19eC21 Briefly, change in concentration of a protein marker (Evan’s blue tagged albumin) instilled into the airspace compartment of isolated and perfused rats lungs is used to calculate change in alveolar volume over a 60-minute experimental period. Fluorescene isothiocyanate (FITC)eCtagged albumin was added to the perfusate to monitor leakage of protein from the vascular compartment into the airspace to produce an index of alveolar permeability.
Rat Treatment Protocols
AFC was measured in sham, adNull, and adCFTR-infected rats (9 animals/group). An additional 5 rats per group were studied with amiloride (10eC6 mol/L) in the alveolar instillate during clearance measurements. To estimate CleC channel function in sham, adNull, and adCFTR-infected rat lungs the CleC transport inhibitors 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 10eC4 mol/L), glibenclamide (10eC4 mol/L), or bumetanide (10eC4 mol/L) were individually added to the alveolar instillate during clearance measurements. The Na+ channel blocker amiloride (10eC6 mol/L) was included in the alveolar instillate in some experiments.
AFC Measurements in Live Mice
The method of measurement used in this study was modified from Hardiman et al and has been described elsewhere.6,22,23 This method is similar to that used in rats except that 300 e蘈 of Evan’s blue-tagged albumin is instilled into lungs of sedated, mechanically ventilated mice that are maintained supine, and clearance is measured over a 30-minute period. Assessment of CleC channel function in mice was via use of a CleC-free instillate solution where NaCl was replaced with equimolar amounts of NaGluconate without altering osmolarity. In some mice, forskolin (10eC4 mol/L) was included in the instillate solution during clearance measurements. Experiments using CleC-free instillate were performed with a positive end-expiratory pressure of 3 cmH2O above ambient pressure using a water valve attached to the expiratory port of a rodent ventilator.
Reverse Transcriptase PCR
Total RNA from peripheral (distal 2 to 3 mm) and proximal (central airways) lung tissue and liver was isolated using RNA-zol B (Tel-Test, Inc). RNA was treated with DNAse I (50 U, Sigma,), twice extracted with phenol-chloroform, precipitated, and resuspended in 20 e蘈 of RNAse-free water before first strand cDNA synthesis using oligo-dT12-16. PCR was performed using 2 e蘈 of the reaction mixture, 30 cycles of amplification, and an annealing temperature of 58°C. Primer sequences used to detect human CFTR mRNA were as follows: upstream 5'ACGCTTCAGGCACGAA and downstream 5'CGGCTACTCCCACGTAA.
Cell Membrane Isolation
Whole-cell membrane proteins and membrane fractions enriched for the basolateral membrane domain were produced from tissue collected from the distal 2 to 3 mm of right rat lungs after serial bronchoalveolar lavage and perfusion of the pulmonary artery with PBS, as previously described.10,16,17,24
Western Analysis
Twenty micrograms of whole-cell membrane protein was separated with 4% to 12% SDS-PAGE (Invitrogen Life Technology), electrophoretically transferred to nitrocellulose and probed with a mouse monoclonal antieChuman CFTR antibody (R&D Systems), a rabbit antieCrat ENaC antibody (Affinity Bioreagents), a rabbit antieCNa,K-ATPase 1 antibody (K. Geering, University of Lausanne, Switzerland)25 or a mouse monoclonal anti-actin antibody (Chemicon International). Protein bands were visualized using peroxidase coupled secondary antibodies and a chemiluminescence detection kit (Pierce).
Na,K-ATPase Function (Pi Liberation From ATP) in the Distal Lung
Twenty micrograms of basolateral cell membrane protein isolated from the peripheral lung (distal 2 to 3 mm) of rats was resuspended in 100 e蘈 of a high [Na+]/low [K+]/high [ATP] reaction buffer (in mmol/L: 50 Tris-HCl pH 7.4, 50 NaCl, 5 KCl, 10 Mg2Cl, 1 EGTA, 10 Na2ATP), with [-32P]-ATP (3.3 e藽i/mL) as previously described.16,17 These conditions maximize Na,K-ATPase activity (Vmax) and produce an index of functional, membrane-bound receptor number. Triplicate samples were placed at eC20°C for 15 minutes before incubation for 15 minutes at 37°C. The reaction was terminated by the addition of 5% TCA/10% charcoal and cooling to 4°C. The charcoal phase containing unhydrolyzed nucleotide was separated by centrifugation (12 000g for 5 minutes) and the liberated 32P quantified. Na,K-ATPase activity was calculated as the difference between the test samples (total/nonspecific ATPase activity) and samples assayed in reaction buffer with 2.5 mmol/L ouabain but devoid of Na+ and K+ (non-Na,K-ATPase ATPase activity). Results are expressed as mean nmol of Pi/mg of protein per hour of triplicate measurements from 3 rats per group.
Immunohistochemistry for CFTR
Mouse lungs (C57b6) were fixed in 2% formaldehyde/0.2% glutaraldehyde imbedded in paraffin and sectioned (5 e). Deparafinized and rehydrated sections were blocked at room temperature for 1 hour with 5% normal goat serum in Superblock before incubation with anti-CFTR antibodies (13-1 and 24-1, R&D Systems, 1:100 dilution; MM13eC4 and M3A7, Upstate USA, Inc, 1:200 dilution) at 37°C for 2 hours and then washed 5 times with PBS. Immunodetection was with Alexa568 (Molecular Probe, 1:1000) for 1 hour (at 37°C) followed by washing 5 times with PBS. The slides were then mounted with Vectashield (Vector Labs, Inc).
Data Analysis
All values are reported as mean±standard deviation (SD). Statistical significance was defined as P<0.05.
Results
Transgene Expression
To produce a model of alveolar CFTR overexpression, a first generation adenovirus that expresses a human CFTR cDNA under the control of a human CMV promoter-enhancer element (adCFTR) was used to transduce the lungs of normal rats (Figure 1). This vector has been shown to increase CFTR function in airway epithelial cells, animal models, and humans with cystic fibrosis.26eC29 Human CFTR mRNA was detected in both proximal and distal rat lung tissue 7 days after infection with adCFTR using rtPCR and human CFTR-specific primers (Figure 1A). No human CFTR mRNA was observed in livers of adCFTR-infected rats or in lungs and livers from adNull-infected controls. Increased CFTR protein expression was noted in cell membranes isolated from the distal lungs of adCFTR-infected rats (Figure 1B). Immunostaining of adCFTR-infected C57b6 mouse lungs using antieChuman CFTR primary antibodies demonstrated patchy immunoreactivity in a linear pattern that surrounded the airspace, consistent with transduction of all alveolar epithelial cells (Figure 1C).
Effects of CFTR Overexpression on AFC in Rats
AFC in rats infected with adCFTR (5x101 viral particles) was 92% greater (0.98±0.11 mL/hour) than adNull (0.51±0.06 mL/hour) and sham-infected controls (0.51±0.02, P<0.002 adCFTR versus sham and adNull) (Figure 2). No flux of albumin from the pulmonary vasculature into the airspace was measurable in any of the experimental groups implying that alveolar barrier function was not altered by gene transfer or CFTR overexpression. To test if CFTR gene transfer affects alveolar CleC channel function, we conducted experiments using 3 established CleC channel inhibitors in the alveolar instillate during clearance measurements. NPPB did not affect AFC in sham- and adNull-infected lungs, whereas glibenclamide and bumetanide reduced it by 20% (Figure 3A through 3C). Glibenclamide, bumetanide, and NPPB reduced AFC in adCFTR-infected lungs by 55% to 60%. Net clearance in adCFTR-infected lungs treated with these agents was the same as similarly treated sham- and adNull-infected controls; thus, CleC channel blockade completely negated the effects of CFTR overexpression.
To further evaluate if CFTR gene transfer alters epithelial CleC channel function, AFC was measured using an instillate solution free of CleC (Figure 3D). Doing so resulted in CleC flux along its concentration gradient from the interstitial into the alveolar airspace and has been shown to cause fluid accumulation in the airspace.4 These experiments were conducted using C57bl6 mice and a positive end-expiratory pressure of 3 cmH2O, which increased basal AFC to 30.29±4.5%/30 minutes. This was substantially greater than that reported previously by our group6 but is consistent with recent data from Hardiman.23 The absence of CleC caused airspace fluid accumulation (AFC: eC8.8±3.7/%30 minutes) that doubled to eC17.5±2.8%/30 minutes when the adenylyl cyclase activator forskolin was included in the instillate of adNull-infected mice. Basal and forskolin-stimulated AFC in adCFTR mice was significantly more negative than adNull (eC19.1±2.7%/30 minutes and eC22.7±5.2%/30 minutes, respectively). These findings are consistent with increased function of a cAMP sensitive CleC channel.
Effects of CFTR Overexpression on Na+ Channel and Na,K-ATPase in Rats
Amiloride (10eC6 mol/L) was included in the alveolar instillate during clearance measurements to assess amiloride-sensitive Na+ channel function. As can be seen in Figure 4A, amiloride reduced AFC to a greater degree in adCFTR-infected lungs than in sham- and adNull-infected controls (AFC: adCFTR, eC57% to 0.42±0.11 mL/hour; adNull, eC46% to 0.26±0.03 mL/hour; sham, eC49% to 0.27±0.07 mL/hr) (P<0.05 adCFTR versus adNull or sham) (Figure 4A). However, the absolute clearance rate in amiloride-treated adCFTR-infected lungs (0.42±0.11 mL/hour) remained greater than similarly treated sham (0.27±0.07 mL/hour) and adNull controls (0.26±0.03 mL/hour) (P<0.03 adCFTR versus sham or adNull). Western analysis for Epithelial Na+ Channel subunit (ENaC) using whole-cell membranes from the peripheral lung did not reveal significant differences in expression between adNull and adCFTR infected lungs (Figure 4B). Thus, CFTR gene transfer increases both amiloride-sensitive and insensitive Na+ channel function in rats. Whether this is due to changes in ENaC expression, the biophysical properties/amiloride sensitivity of ENaC, or upregulation of an amiloride-insensitive apical Na+ entry pathway is not yet known.
The effect of CFTR gene transfer on Na,K-ATPase activity was assessed by measuring ouabain-sensitive liberation of inorganic phosphate (Pi) from ATP in the presence of substrate independent conditions (high [Na+]/low [K+]/high [ATP]) that allow the Na+ pump to function maximally (Vmax). Under these conditions, the determinate of Pi release is the number of functional Na,K-ATPases in basolateral membranes from the peripheral lung. AdCFTR-infected rat lungs had up to 66% more functional Na,K-ATPases in basolateral membranes than sham- and adNull-infected controls (2.91±0.32, 1.57±0.23, and 1.75±0.42 nmol Pi/mg protein per hour, respectively; P<0.02 adCFTR versus sham and adNull) (Figure 5A). Western analysis likewise showed increased expression of the Na,K-ATPase 1 subunit in adCFTR-infected lungs (Figure 5B). These findings indicate that upregulation of CleC channel function results in parallel changes of other transport proteins.
CFTR Gene Transfer to Alveoli of Mice With Targeted Deletions of the 1- and 2-Adrenergic Receptors
We have recently reported that mice with targeted deletions of the 1 and 2 adrenergic receptors (1AReC/eC/2AReC/eC) have decreased basal AFC, Na,K-ATPase function, and amiloride sensitivity.6 Rescue of alveolar epithelial 2AR function normalizes these processes confirming that these mice retain G-protein signaling capability and can upregulate lung ion transport proteins. To test if changes in active transport after CFTR gene transfer require 2AR function, we infected mice with 1x1010 viral particles of adCFTR 7 days before study using a recently described surfactant-based delivery system.6 Fluid clearance in this model was measured using a "live" mechanically ventilated intact lung model that preserves oxygenation, ventilation, and serum pH.22,23 CFTR gene transfer increased AFC in strain-specific 1AR+/+/2AR+/+ mice by 36% (from 22.2±3.0% to 30.3±2.9%; P<0.02 versus uninfected, sham, and adNull infected 1AR+/+/2AR+/+’; Figure 6A). This increase was less than that noted in rats (Figure 2) and is likely due to lower transduction efficiency (70%6 versus >90%10,14) and perhaps decreased dependence on CFTR for AFC in mice than rats. Importantly, CFTR gene transfer to the alveolar epithelium of 1AReC/eC/2AReC/eC mice had no effect on AFC measured 7 days after gene transfer. AdNull infection did not affect AFC at this postinfection time-point in either the 1AR+/+/2AR+/+ or 1AReC/eC/2AReC/eC groups. Clearance in these groups was the same as untreated/uninfected mice in a prior study.2
2AR Gene Transfer to the Alveoli of Mice With a Nonfunctional Human CFTR
To further approach the question of a functional interaction between 2AR and CFTR, we transduced the alveolar epithelium of mice that have no mouse CFTR gene (CFTReC/eC) and are homozygous for a nonfunctional human CFTR gene (508 mutation) with an adenovirus that expresses a human 2AR gene (ad2AR) (Figure 6B). We have shown that this approach increases 2AR expression only in the alveolar epithelium and upregulates 2AR function and alveolar active Na+ transport in both wild-type and 1AReC/eC/2AReC/eC mice.6 Increased alveolar -receptor function in this model appears to be due to increased numbers of 2ARs in the cell membrane and enhanced sensitivity to endogenous catecholamines. As can be seen in Figure 6B, 2AR gene transfer did not affect AFC in CFTReC/eC mice but increased it by 42% in wild-type controls (from 21.7±4.2% to 30.7±5.6%; P=0.04 versus sham-infected CFTR+/+). Clearance in sham- and adNull-nfected CFTReC/eC mice was similar to sham (Figure 6B) and uninfected2 wild-type mice consistent with other reports showing that CFTR may not be required for maintenance of basal alveolar active Na+ transport.30 These data, combined with that from experiments of CFTR gene transfer to 1AReC/eC/2AReC/eC mice suggest that 2AR mediates increases in alveolar active Na+ transport via a CFTR-dependent pathway and that CFTR function requires the presence of the 2AR.
Discussion
In the current study, we tested if upregulation of CFTR function, via adenoviral-mediated overexpression, alters alveolar active Na+ transport in rodents. To affect this goal, we used previously tested adenovectors and established delivery strategies that produce transgene expression in both type 1 and 2 alveolar epithelial cells in rats and mice (Figure 1C).16 Data from other groups indicates that the 2AR is present in both type 1 and 2 epithelial cells,30,31 thus it is likely that the methods used in these experiments result in concomitant expression of CFTR and 2AR in both types of alveolar epithelial cells.
The functional effects of CFTR gene transfer in rats were assessed by measuring the capacity of the lung to clear fluid instilled into the lung using a well-described isolated rat lung model.10 This experimental design is based on the principal that AFC is an index of active, vectorial Na+ transport by alveolar epithelial cells. Using this approach, we observed that overexpression of a human CFTR increases alveolar active Na+ transport by nearly 100% in normal rats (Figure 2) and by 42% in mice (Figure 6B). Inclusion of CleC channel blockers in the alveolar instillate during clearance measurements obviated the effects of CFTR overexpression (Figure 3). None of the blocking agents used is perfectly specific for CFTR, thus additional studies were conducted in mice using a CleC-free instillate. This model results in CleC and fluid flux into the airspace and can be used to assess CleC channel function.4 Using this method, we noted that airspace fluid accumulation was greater in adCFTR-infected mice than in untreated and forskolin-treated adNull controls (Figure 3D). The inclusion of the adenylyl cyclase activator forskolin in the airspace of adCFTR-infected mice produced significant incremental fluid accumulation consistent with increased function of a cAMP-regulated CleC channel such as CFTR.
To probe how CFTR overexpression effects Na+ channel function, we included amiloride in the alveolar compartment during clearance measurements in rats (Figure 4A). Amiloride reduced alveolar active Na+ transport in lungs overexpressing CFTR, implying that increased CFTR function results in increased amiloride-sensitive Na+ channel (ie, epithelial Na+ channel) function (Figure 4A). Absolute clearance rates in amiloride-treated adCFTR lungs were greater than in similarly treated controls, suggesting that CFTR gene transfer also upregulates amiloride-insensitive Na+ entry pathways (Figure 4A). CFTR-infected rats also had significant increases in the number of functional Na,K-ATPases in basolateral cell membranes (Figure 4B). How then does CFTR overexpression increase vectorial ion transport We speculate that overexpression of CFTR increases the number of open/activated CFTRs in the cell membraneeCenhancing CleC entry and results in upregulation of amiloride-sensitive and insensitive Na+ entry pathways. This hypothesis requires that CFTR activate ENaC and other Na+ entry pathways and is in contradistinction to data from MDCK cells.32 Importantly, our data suggests that the interaction of CFTR with apical Na+ channels in alveolar epithelial cells might be markedly different than in proximal airway epithelial cells. Irrespective of this speculation, increased Na+ flux in the setting of CFTR overexpression results in upregulation of basolateral Na,K-ATPase capacity. The net result of these effects is increased vectorial Na+ flux and accelerated AFC.
Both 1- and 2-adrenergic agonists upregulate alveolar active Na+ transport in experimental models16 and humans.33,34 These agonists increase trafficking of Na,K-ATPases to the basolateral cell membrane, cause trafficking to the cell membrane and opening of epithelial Na+ channels, and increase CleC flux through CFTR.2 To probe for a functional interaction between alveolar AR and CFTR, we tested the effects of CFTR overexpression in mice with targeted deletions of both the 1- and 2- adrenergic receptor (1AReC/eC/2AReC/eC). We have previously shown that these mice have normal total lung cAMP levels and retain G-protein signaling and adenylyl cyclase function but have much reduced basal alveolar active Na+ transport rates that can be normalized via rescue of 2AR function into the alveolar epithelium.6 CFTR overexpression in 1AReC/eC/2AReC/eC mice had no effect on alveolar active Na+ transport (Figure 6A). Thus, CFTR overexpression, in and of itself, does not alter vectorial ion flux across the alveolar epithelium. This observation caused us to test the effect of 2AR gene transfer to the alveolar epithelium of 508 mice. These crucial studies revealed that 2AR gene transfer in the absence of functional CFTR does not accelerate alveolar active Na+ transport as it does in wild-type controls. Recently, Fang and colleagues35 reported that CFTReC/eC mice are unable to compensate for increased lung water in a model of hydrostatic pulmonary edema and, importantly, do not accelerate alveolar active Na+ transport in response to -agonist treatment. We believe that the data from the present study and that of Fang support a new paradigm where CFTR and the AR are functionally interdependent and both are required to upregulate alveolar active Na+ transport in the setting of pathological increases of fluid in the alveolar airspace. Presumptive mechanisms for this interaction have been suggested by Naren and colleagues who demonstrated that in airway epithelial cells (calu-3) 2ARs physically associate with CFTR through the PDZ domain of ezrin/radixin/moesin-binding phosphoprotein 50.36 The formation of this macromolecular complex facilitates access of cAMP-activated protein kinase to R domain sites on CFTR and is required for cAMP-mediated regulation of CFTR.
The findings of this study raise the possibility that CleC transport may be a rate-limiting step for vectorial ion transport in the alveolar epithelium. While possible, we believe that alveolar active transport requires several key transport proteins (eg, ENaC, CFTR, Na,K-ATPase) that must interact to generate a transepithelial NaCl gradient, and that impairment of any of these protein families will impede AFC. Likewise, based on data from other studies, upregulation of single member of the transport pathway results in compensatory augmentation of other pathway components.6,10,11,16eC18
The present study provides new evidence that CFTR is an important contributor to the pathway by which the lung clears fluid from the alveolar airspace and that there exists an interdependence between 2AR and CFTR where both are required to upregulate alveolar active Na+ transport. These studies raise concern that the alveolar epithelium of patients with cystic fibrosis may lack the capacity to upregulate alveolar active Na+ transport in response to lung injury. The results of this study also suggest that upregulation of alveolar CFTR function might be an adjunct for improving the lung’s ability to clear pulmonary edema.
Acknowledgments
This work was supported by HL-66211, HL-71042, HL-19094, American Heart Association, and the Evanston Northwestern Healthcare Research Institute. We thank Dr K. Geering (Lausanne University) for providing us with antieCNa,K-ATPase 1 antibody.
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