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(Circulation Research. 2004;94:1091.)
© 2004 American Heart Association, Inc.

Cellular Biology

Upregulation of Alveolar Epithelial Active Na⁺ Transport Is Dependent on ß₂-Adrenergic Receptor Signaling

Gökhan M. Mutlu, Vidas Dumasius, James Burhop, Pamela J. McShane, Fan Jing Meng, Lynn Welch, Andrew Dumasius, Nima Mohebahmadi, Gloria Thakuria, Karen Hardiman, Sadis Matalon, Steven Hollenberg, Phillip Factor

From the Division of Pulmonary and Critical Care Medicine (G.M.M., L.W.), Northwestern University Feinberg School of Medicine, Chicago, Ill; University of Illinois College of Medicine (V.D.), Chicago, Ill; Evanston Northwestern Healthcare Research Institute (J.B., F.J.M., N.M.), Evanston, Ill; Division of Pulmonary and Critical Care Medicine (P.J.M.), University of Rochester, Rochester, NY; Rush Presbyterian St Lukes Hospital (A.D.), Chicago, Ill; Department of Anesthesiology (K.H., S.M.), University of Alabama at Birmingham, Birmingham, Ala; Section of Cardiology (S.H.), Cooper Hospital/University Medical Center, Camden, NJ; Division of Pulmonary (P.F.), Allergy and Critical Care Medicine, Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to Phillip Factor, DO, Pulmonary, Allergy and Critical Care Medicine, Columbia University College of Physicians and Surgeons, P&S 10-502, 630 W 168th St, New York, NY 10032. E-mail phf2103{at}columbia.edu

	Abstract

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Alveolar epithelial ß-adrenergic receptor (ßAR) activation accelerates active Na⁺ transport in lung epithelial cells in vitro and speeds alveolar edema resolution in human lung tissue and normal and injured animal lungs. Whether these receptors are essential for alveolar fluid clearance (AFC) or if other mechanisms are sufficient to regulate active transport is unknown. In this study, we report that mice with no ß₁- or ß₂-adrenergic receptors (ß₁AR^–/–/ß₂AR^–/–) have reduced distal lung Na,K-ATPase function and diminished basal and amiloride-sensitive AFC. Total lung water content in these animals was not different from wild-type controls, suggesting that ßAR signaling may not be required for alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with ß₁- but not ß₂-adrenergic receptors to ß₁AR^–/–/ß₂AR^–/– mice indicates that the ß₂AR mediates the bulk of ß-adrenergic-sensitive alveolar active Na⁺ transport. To test the necessity of ßAR signaling in acute lung injury, ß₁AR^–/–/ß₂AR^–/–, ß₁AR^+/+/ß₂AR^–/–, and ß₁AR^+/+/ß₂AR^+/+ mice were exposed to 100% oxygen for up to 204 hours. ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice had more lung water and worse survival from this form of acute lung injury than wild-type controls. Adenoviral-mediated rescue of ß₂-adrenergic receptor (ß₂AR) function into the alveolar epithelium of ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice normalized distal lung ß₂AR function, alveolar epithelial active Na⁺ transport, and survival from hyperoxia. These findings indicate that ßAR signaling may not be necessary for basal AFC, and that ß₂AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace of normal and injured lungs.

Key Words: alveolar fluid clearance • pulmonary edema • ß₂-adrenergic receptor • adenovirus • Na⁺ channel

	Introduction

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The combined action of alveolar epithelial Na⁺ channels (ENaCs), the cystic fibrosis transmembrane conductance regulator (CFTR), Na,K-ATPases, and K⁺ channels creates the transepithelial Na⁺ gradient needed for the transit of excess fluid from the alveolar airspace.^1,2 The importance of these proteins to this energy-dependent (ie, active) process is evidenced by data showing that their inhibition reduces the lung’s ability to clear excess alveolar fluid^3–7 and that their upregulation confers protection from acute injury.^4,8,9 Despite these extensive investigations, the mechanisms by which these proteins are upregulated in response to excess alveolar fluid (pulmonary edema) are not well resolved.

One possible pathway for upregulation of alveolar-active Na⁺ transport is ß-adrenergic receptor activation. Stimulation of alveolar epithelial ßARs by endogenous or exogenous catecholamines accelerates active Na⁺ transport in lung epithelial cells in vitro and in experimental in vivo systems by increasing the expression and/or function of epithelial transport proteins.^10–12 Thus, this G protein-dependent pathway represents a mechanism by which the lung can alter its physiology to adapt to and protect itself from excess alveolar fluid. What is not known is if ßAR signaling is essential for the regulation of alveolar active Na⁺ transport or whether other mechanisms (eg, intracellular osmo-, redox-, or chemo-sensitive regulators) can enhance alveolar active transport to clear pulmonary edema.

The present study was structured to define what contribution alveolar epithelial ßARs make to active Na⁺ transport in the alveolar epithelium of normal mice and mice with acute lung injury caused by exposure to hyperoxia. Herein, we show that distal lung transport protein function and the lung’s ability to clear excess alveolar fluid is highly dependent on alveolar epithelial ß₂-adrenergic receptor (ß₂AR) function and that the absence of alveolar ß₂-receptor function compromises survival from an acute lung injury.

	Materials and Methods

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Adenovirus Propagation and Purification
Replication-incompetent E1a^–/E3^– adenoviruses containing a human CMV driven human ß₂AR cDNA (adß₂AR, a gift of Drs Robert Lefkowitz and Walter Koch, Duke University), an Escherichia coli lac Z gene (adß-gal), or no cDNA (adNull) were propagated, purified, and titered as previously described.^13,14

Animals
The use of animals for this study was approved by the Evanston Northwestern Healthcare Institutional Animal Use and Care Committee. Specific pathogen-free adult male C57BL/6 mice were from Harlan (Indianapolis, Ind). Mice with targeted deletions of the ß₂AR (ß₁AR^+/+/ß₂AR^–/–), both ß₁AR and ß₂AR genes (ß₁AR^–/–/ß₂AR^–/–), and strain-specific ß₁AR^+/+/ß₂AR^+/+ (wild-type) mice, were from Dr Brian Kobilka (Stanford University, Calif).^15,16

Adenovirus Delivery to Mouse Lungs
Mice were anesthetized with pentobarbital (75 mg/kg, IP) and orally intubated. Adenovirus, in 25 µL of 100% surfactant (Survanta, Abbott LaboratoriesL), followed by 200 µL of air was administered via the endotracheal tube.¹⁴ A second instillation was performed 5 minutes after the first. All adenovirus-infected animals received 1x10¹¹ viral particles 7 days before study. The distribution of gene transfer using this method was assessed by infecting mice (n=4) with adß-gal and X-gal staining as previously described.¹⁴

Alveolar Fluid Clearance (AFC) Measurement
The method used to quantify the rate of removal of fluid from the alveolar airspace (alveolar fluid clearance) was from Hardiman¹⁷ except that mice were maintained supine. Alveolar fluid clearance was calculated based on the change in concentration of Evan’s blue tagged albumin in an isoosmolar (324mOsm) alveolar instillate placed into the alveolar airspace over a 30-minute period of measurement. In some experiments procaterol (a specific ß₂AR agonist, 10^–8 mol/L) or amiloride (10^–3 mol/L) were administered in the instillate. Amiloride sensitivity is reported as percent reduction AFC as compared with similarly treated mice not exposed to amiloride.

Immunohistochemistry
Longitudinal sections (3 µm) of left lungs fixed with 4% paraformaldehyde were treated with 3% H₂O₂ before blocking of nonspecific immunoreactivity with nonimmune goat serum. Rabbit anti-human ß₂AR antibody (1:500 dilution, Santa Cruz Scientific) and a fluorescein-linked secondary antibody (Vector Elite ABC kit, Vector Laboratories) were used for immunodetection.

Whole and Basolateral Cell Membrane Isolation and Western Analysis
Membrane proteins were obtained by homogenizing lung tissue collected from the peripheral 1 to 2 mm of each lobe and used for Western analysis using an anti-rat ß₂AR antibody (Santa Cruz Scientific) as described previously and in the expanded Materials and Methods section in the online data supplement (http://circres.ahajournals.org) to this study.^11,18

Measurement of cAMP Levels
Cyclic-AMP production by whole-cell membrane fractions (5 to 10 µg) from peripheral lung tissue over 30 minutes was measured using a radioimmunoassay (Amprep SAX, NEN/Perkin Elmer) as described previously.¹¹

Na,K-ATPase Function (P_i Liberation From ATP) in the Distal Lung
Na,K-ATPase activity was quantified by comparing the amount of inorganic phosphate (P_i) liberated from ATP over 1 hour by 20 µg of basolateral cell membrane protein isolated from the peripheral lung in the presence and absence of the Na,K-ATPase inhibitor ouabain under conditions that maximize Na,K-ATPase activity (V_max) as previously described^11,18 and in the online data supplement.

Echocardiographic Assessment of Cardiac Function
Parasternal long and short axis M-mode echocardiographic images from lightly sedated mice were used to obtain average left ventricular end-diastolic (LVEDd) dimensions. Aortic outflow tract diameter was determined in the parasternal long axis by M-mode. Continuous wave Doppler was used to measure aortic outflow tract velocities in an apical 4-chamber view. Stroke volume was calculated by multiplying aortic area by the time-velocity integral of aortic outflow. Cardiac output was calculated by multiplying stroke volume by heart rate.¹⁹

Induction of Acute Lung Injury
Mice were exposed to hyperoxia (100% normobaric O₂) in two sets of experiments. In one set of experiments, mice were exposed for 66 hours before measurement of total lung water content (lung wet-dry weight ratio) as previously described (n=3 mice/group).²⁰ In the second set, survival studies were conducted by exposure for up to 204 hours. Studies of adenovirus-infected animals hyperoxia were initiated 7 days after infection. Surviving animals were enumerated at 12-hour intervals (n=6 mice/group).

Data Analysis
All values are reported as mean±SD. Statistical comparison among groups was performed using one-way ANOVA (GraphPad Prism, GraphPad Software, Inc). Comparison of survival among groups was performed using a Kaplan-Meier method to determine the LD₅₀ (Graphpad Prism). Statistical significance in all experiments was defined as P<0.05.

	Results

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Alveolar Active Na⁺ Transport Is Reduced in Mice With Targeted Deletions of the ß₁AR and/or ß₂AR
The clearance of edema fluid from the alveolar airspace is a consequence of active extrusion of Na⁺ from the airspace into the interstitium by alveolar epithelial cells (Figure 1). Thus, alveolar fluid clearance (AFC) rate can be used as an index of active Na⁺ transport in the alveolar epithelium. To determine what contribution ßARs make to this process, AFC was measured in mice with targeted deletions of the ß₂AR (ß₁AR^+/+/ß₂AR^–/–) and both ß₁AR and ß₂AR (ß₁AR^–/–/ß₂AR^–/–) using a modification of the mechanically ventilated, intact lung model described by Hardiman and colleagues.¹⁷ In the present experiments, mice were maintained supine, which results in distribution of the 300 µL of alveolar instillate to both lungs. Preliminary studies indicated that AFC is proportional to the volume of fluid instilled, however, animal mortality increases with volumes in excess of 300 µL. Thus, this change in position accounts for the lower AFC rates in this study than reported by Hardiman. Using this method, we measured AFC rates of 21.9±4.0%/30 minutes (n=4) in strain-specific ß₁AR^+/+/ß₂AR^+/+ (wild-type) and 22.2±3.0%/30 minutes (n=15) in C57BL/6 ß₁AR^+/+/ß₂AR^+/+ control mice (Figure 1A). Importantly, AFC in mice with ß₁- but no ß₂AR function (ß₁AR^+/+/ß₂AR^–/–) and mice with no ß₁- or ß₂AR function (ß₁AR^–/–/ß₂AR^–/–) was unto 44% less than in ß₁AR^+/+/ß₂AR^+/+ controls (ß₁AR^+/+/ß₂AR^–/–, 15.2±2.4%/30 minutes, n=4; ß₁AR^–/–/ß₂AR^–/–, 12.2±5.2%/30 minutes, n=6; P<0.01 ß₁AR^–/–/ß₂AR^–/– or ß₁AR^+/+/ß₂AR^–/– versus wild-type and C57BL/6). In all experiments, the volume of fluid aspirated from the lungs at the conclusion of AFC measurements in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice was greater than from ß₂AR^+/+/ß₁AR^+/+ controls (100 versus 50 µL). The inclusion of the ß₂AR specific agonist procaterol (10^–8 mol/L) in the alveolar instillate solution increased clearance in ß₁AR^+/+/ß₂AR^+/+ wild-type mice by 50% from 21.9±4.0% per 30 minutes to 30.1±1.3% per 30 minutes (n=4), but, as expected, had no effect in ß₁AR^–/–/ß₂AR^–/– mice (11.6±3.1% per 30 minutes, n=4) or ß₁AR^+/+/ß₂AR^–/– mice (15.0±1.5% per 30 minutes, n=4) (Figure 1B). The reduced basal AFC rates in the ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice confirm an important role for ß₂AR signaling in the regulation of alveolar epithelial active Na⁺ transport in mice.

View larger version (32K): [in this window] [in a new window]   Figure 1. Alveolar fluid clearance in mice. A, Alveolar fluid clearance in uninfected ß1AR+/+/ß2AR+/+ C57BL/6 and wild-type mice and ß1AR+/+/ß2AR–/– and ß1AR–/–/ß2AR–/– mice in the presence and absence of the ß2AR-specific agonist procaterol in the alveolar instillate fluid during clearance measurements (, untreated; , procaterol). *P<0.05 ß1AR+/+/ß2AR–/– or ß1AR–/–/ß2AR–/– mice vs untreated wild-type and untreated C57BL/6. **P<0.05 vs untreated same group. B, Alveolar fluid clearance in uninfected, sham-, adNull-, and adß2AR-infected ß1AR+/+/ß2AR+/+, ß1AR+/+/ß2AR–/–, and ß1AR–/–/ß2AR–/– mice. *P<0.05 adß2AR-infected vs uninfected, sham-, and adNull-infected ß1AR+/+/ß2AR+/+ mice; **P<0.05 uninfected, sham-, and adNull-infected ß1AR–/–/ß2AR–/– mice vs adß2AR-infected ß1AR–/–/ß2AR–/– mice and all groups in ß1AR+/+/ß2AR+/+ mice. C, Effect of inclusion of the ß2AR specific agonist procaterol in the alveolar instillate of sham- and adß2AR-infected ß1AR+/+/ß2AR+/+ and ß1AR–/–/ß2AR–/– mice (, untreated; , procaterol). *P<0.05 procaterol-treated vs untreated, sham-infected ß1AR+/+/ß2AR+/+ mice; **P<0.05 untreated and procaterol-treated ß1AR–/–/ß2AR–/– mice vs all other groups. D, Changes in AFC after isoproterenol administration in ß1AR+/+/ß2AR+/+ and ß1AR+/+/ß2AR–/– mice (, untreated; , isoproterenol). *P<0.05 vs untreated ß1AR+/+/ß2AR+/+.

ß₂AR Gene Transfer Normalizes Alveolar Active Na⁺ Transport in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– Mice
To assess the contribution of ßAR function to active Na⁺ transport in the alveolar epithelium, ß₁AR^–/–/ß₂AR^–/–, ß₁AR^+/+/ß₂AR^–/–, and ß₂AR^+/+/ß₁AR^+/+ mice were infected with a replication-incompetent, E1a^–/E3^– recombinant adenovirus that expresses a human ß₂AR (adß₂AR) under the control of a human CMV promoter-enhancer element (Figure 1B). The surfactant-based delivery method we used to transduce the alveolar epithelium is based on prior studies in rats and yielded transgene expression in all lung lobes (Figure 2). Quantification of gene transfer using a linear intercept method in mice infected with adß-gal indicated that 68±5% and 70±7% of alveoli in ß₁AR^+/+/ß₂AR^+/+ and ß₁AR^–/–/ß₂AR^–/– mice, respectively, had evidence of transgene expression (Figure 2A). Immunostaining of lungs of adß₂AR infected mice for human ß₂AR produced a linear pattern of immunoreactivity that extends all, or much, of the way around the airspace of many alveoli suggesting that adenoviral vectors transduce type 1 and type 2 alveolar epithelial cells (Figure 2C). In all gene transfer experiments, mice were studied 7 days after infection, to allow vector-induced host responses to subside. To further control for the effects of adenovirus-induced inflammation on alveolar active Na⁺ transport all experiments included control animals infected with adNull. Rescue of alveolar ß₂AR function into ß₁AR^–/–/ß₂AR^–/– mice with adß₂AR increased AFC by 77% from 12.2±5.2% to 21.6±4.1% per 30 minutes (n=4), and by 88% from 15.2±2.4% to 26.6±1.5% per 30 minutes in ß₁AR^+/+/ß₂AR^–/– mice (n=4). These rates of AFC were not different from uninfected ß₁AR^+/+/ß₂AR^+/+ C57BL/6 or wild-type mice, although the magnitude of increase in ß₁AR^+/+/ß₂AR^–/– was slightly greater than ß₁AR^–/–/ß₂AR^–/– (P=0.045). ß₂AR gene transfer also increased clearance in strain-specific wild-type (ß₁AR^+/+/ß₂AR^+/+) mice (44.7% to 31.7.1±4.4%/30 minutes, n=4) (Figure 1B). Sham and adNull infection did not affect AFC in the ß₁AR^–/–/ß₂AR^–/–, ß₁AR^+/+/ß₂AR^–/–, or ß₁AR^+/+/ß₂AR^+/+ groups. Thus, ß₂AR function in 70% of alveoli is sufficient to normalize AFC in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice. These experiments are the first to both localize transgene expression and demonstrate a relevant physiological effect after adenoviral-mediated gene transfer in the distal lung of mice.

View larger version (65K): [in this window] [in a new window]   Figure 2. Transgene expression in mouse lungs. A, Representative lungs from a ß1AR+/+/ß2AR+/+ (wild-type) mouse infected with 1x1011 viral particles of adß-gal and stained with X-gal 7 days later. Adjacent photomicrographs are of 3-µm sections of paraffin-imbedded lungs from ß1AR+/+/ß2AR+/+ and ß1AR–/–/ß2AR–/– mice (original magnification 40x). Transfection efficiency was enumerated in 3 mice/group as the number of alveoli with at least 1 cell with perinuclear ß-galactosidase activity. Transfection efficiency was 70±12% in ß1AR+/+/ß2AR+/+ mice and 68±14% in ß1AR–/–/ß2AR–/– mice (P=NS). B, Representative Western blot of whole cell membrane fractions from peripheral lungs of ß1AR+/+/ß2AR+/+ mice infected with vehicle (sham), adNull, or adß2AR showing the presence of a human ß2AR only in lungs infected with adß2AR. C, Immunostaining of whole lungs using an anti-human ß2AR antibody showing the presence of a human ß2AR only in the alveoli of adß2AR-infected animals. Circumferential pattern of immunostaining is consistent with transduction of both alveolar type 1 and type 2 epithelial cells. Original magnification: 400x.

Inclusion of the specific ß₂-agonist procaterol in the isotonic alveolar instillate fluid had no effect on AFC in ß₁AR^+/+/ß₂AR^+/+ or ß₁AR^–/–/ß₂AR^–/– mice after infection with adß₂AR (32.4±1.3% per 30 minutes, n=3 and 20.8±4.1% per 30 minutes, n=3, respectively) (Figure 1C). This finding is consistent with recent work showing that receptor overexpression maximally upregulates ß₂AR-sensitive AFC without the addition of exogenous catecholamines in rats, and that the human ß₂AR cDNA used in this study is not constitutively active.¹¹ Prior studies in adrenalectomized rodents suggests that increased ßAR function after ß₂AR gene transfer is due to increased numbers of receptors in the cell membrane and possibly enhanced sensitivity to endogenous catecholamines.

ß₁AR Signaling Does Not Contribute to Alveolar Active Na⁺ Transport to the Same Degree as ß₂ARs
The contribution of ß₁AR signaling to basal AFC was measured by including the nonspecific ß-agonist isoproterenol (10^–4 mol/L) in the alveolar instillate of ß₁AR^+/+/ß₂AR^–/– mice (Figure 1D). Isoproterenol stimulation increased AFC in these mice by 24% to 18.9±2.3%/30, a level which was less than in untreated ß₁AR^+/+/ß₂AR^+/+ controls (21.9±4.0% per 30 minutes). Isoproterenol increased clearance by 41% (to 30.9±2.3% per 30 minutes) in strain-specific ß₁AR^+/+/ß₂AR^+/+ mice (P=0.001 isoproterenol treated ß₁AR^+/+/ß₂AR^–/– versus isoproterenol-treated ß₁AR^+/+/ß₂AR^+/+). The greater degree of change in these mice is probably due to isoproterenol-mediated activation of ß₂ARs. Importantly, basal clearance in untreated ß₁AR^+/+/ß₂AR^–/– was not statistically different from that of untreated ß₁AR^–/–/ß₂AR^–/– mice (P=0.35) (Figure 1A). These data provide evidence that ß₁ARs do not contribute to basal AFC to the same degree as ß₂ARs in normal mice. The use of ßAR knockout mice in these studies is particularly relevant as the absence of the ß₂AR might allow for compensatory expansion of the role of the ß₁AR in regulating AFC in ß₁AR^+/+/ß₂AR^–/– mice, hence the modest changes in AFC in isoproterenol treated ß₁AR^+/+/ß₂AR^–/– mice may overstate the contribution of the ß₁AR to AFC in wild-type mice.

ß₂AR Rescue Normalizes Peripheral Lung ß-Receptor Function in ß₁AR^–/–/ß₂AR^–/– Mice
Baseline cAMP production by whole-cell membranes from the peripheral lungs of sham-infected ß₁AR^–/–/ß₂AR^–/– mice was 33% of that from sham-infected wild-type mice (P=0.04, n=3 mice/group) (Figure 3). These membranes did not respond to the ß₂AR agonist procaterol (10^–8 mol/Lx30 minutes) (Figure 3A). Infection of ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^+/+ mice with adß₂AR had no significant effect on basal cAMP production.

View larger version (31K): [in this window] [in a new window]   Figure 3. A, ß-receptor function in peripheral lung membranes. Baseline (untreated), procaterol-responsive, and forskolin-induced cAMP production by whole-cell membrane fractions isolated from peripheral lung tissue (, untreated; , procaterol; , forskolin). *P<0.05 vs same treatment group ß1AR+/+/ß2AR+/+ mice. B, Cyclic-AMP content in peripheral lung tissue homogenates.

Procaterol-induced cAMP production (an index of ß₂AR function) by cell membranes from adß₂AR-infected ß₁AR^–/–/ß₂AR^–/– mice was 5.73±0.4 pmol/mg protein, which is similar to that in sham-infected ß₁AR^+/+/ß₂AR^+/+ mice (3.8±0.4 pmol/mg protein) but significantly less than adß₂AR-infected ß₁AR^+/+/ß₂AR^+/+ mice (10.7±1.4 pmol/mg protein). Thus, ß₂AR gene transfer rescues normal ß₂AR function in the distal lung of ß₁AR^–/–/ß₂AR^–/– mice and results in increased receptor function in wild-types. A small increase in procaterol-sensitive cAMP production by membranes from adNull-infected ß₁AR^+/+/ß₂AR^+/+ mice was noted and was similar to nonspecific changes caused by viral infection in a prior study in rats.¹¹ Interestingly, lung tissue cAMP content measured in distal lung homogenates from ß₁AR^–/–/ß₂AR^–/– mice was not different from strain-specific ß₁AR^+/+/ß₂AR^+/+ mice (Figure 3B). Thus, low intracellular cAMP is not the explanation for the reduced active Na⁺ transport noted in the ß₁AR^–/–/ß₂AR^–/– mice.

Forskolin-induced cAMP production, an index of adenylyl cyclase function, was lower in all ß₁AR^–/–/ß₂AR^–/– groups (Figure 3A). ß₂AR gene transfer increased forskolin-induced cAMP production in both wild-type and knockout mice infected with adß₂AR. These findings suggest that distal lung ßARs may participate in the regulation of their downstream signaling pathways or that the absence of basal/tonic signaling influences of the ß₂AR on adenylyl cyclase could result in its downregulation. Nevertheless, membranes from ß₁AR^–/–/ß₂AR^–/– mice retained responsiveness to forskolin, indicating that signaling systems downstream from the ß₂AR are preserved.

ß-Receptor Function Is Required for Normal Amiloride-Sensitive Alveolar Fluid Clearance (AFC) and Distal Lung Na,K-ATPase Activity
To probe why active Na⁺ transport is diminished in ß₁AR^–/–/ß₂AR^–/– mice, the function of two key alveolar transport proteins was evaluated (Figure 4). An indirect index of epithelial Na⁺ channel function was generated by comparing AFC measured with the Na⁺ channel blocker amiloride (10^–3 mol/L) in the alveolar instillate to that of mice without amiloride. Amiloride reduced AFC by 17% and 16% in sham and adNull-infected ß₁AR^–/–/ß₂AR^–/– mice, respectively, which was significantly less than the reduction noted in sham and adNull-infected ß₁AR^+/+/ß₂AR^+/+ mice (50 and 45%, respectively; P<0.05 sham or adNull infected ß₁AR^–/–/ß₂AR^–/– versus sham or adNull-infected ß₁AR^+/+/ß₂AR^+/+) and is suggestive of diminished amiloride-sensitive Na⁺ transporter function. Rescue of ß₂AR function into the alveolar epithelium of ß₁AR^–/–/ß₂AR^–/– mice restored amiloride-sensitivity to nearly normal (44% reduction of AFC; P=0.45 versus ß₁AR^+/+/ß₂AR^+/+). Amiloride decreased AFC to a greater degree in adß₂AR-infected, ß₁AR^+/+/ß₂AR^+/+ animals (59% to 13.0±3.7%/30 minutes, n=4) than in shams, indicating that ß₂AR overexpression upregulates amiloride-sensitive Na⁺ channel function. Minakata and colleagues²¹ have reported that treatment of isolated rat alveolar type 2 epithelial cells with propranolol for 2 days decreases expression of the epithelial Na⁺ channel -subunit. These prior data and the current results indicate that normal alveolar Na⁺ channel function requires ßAR signaling.

View larger version (24K): [in this window] [in a new window]   Figure 4. A, Effect of amiloride on AFC. Data are percent reduction AFC (as compared with untreated controls) in ß1AR+/+/ß2AR+/+ and ß1AR–/–/ß2AR–/– mice. *P<0.05 adß2AR-infected vs sham- and adNull-infected ß1AR+/+/ß2AR+/+ mice; **P<0.05 sham- and adNull-infected ß1AR–/–/ß2AR–/– mice vs all groups. B, Na,K-ATPase activity (ouabain sensitive liberation of Pi from ATP) in basolateral cell membranes isolated from the peripheral lung. *P<0.05 vs sham-infected ß1AR+/+/ß2AR+/+ mice.

The impact of ßAR signaling on Na,K-ATPase function was assessed by measuring Na,K-ATPase activity (ouabain-sensitive liberation of P_i from ATP) by basolateral membranes isolated from the peripheral lung. Na,K-ATPase activity in sham and adNull-infected mice ß₁AR^–/–/ß₂AR^–/– was 30% of that in similarly infected ß₁AR^+/+/ß₂AR^+/+ mice (P<0.02 sham or adNull ß₁AR^–/–/ß₂AR^–/– mice versus sham or adNull ß₁AR^+/+/ß₂AR^+/+ mice) (Figure 4B). Restoration of ßAR function in the alveolar epithelium with adß₂AR increased maximal Na,K-ATPase activity more than 20-fold to a level similar to adß₂AR-infected ß₁AR^+/+/ß₂AR^+/+ mice. The assay used to measure Na,K-ATPase activity is performed in the presence of high [ATP], high [Na⁺], and low [K⁺]. These "substrate independent" conditions allow the enzyme to function maximally, thereby producing an indirect index of the number of functional enzymes in the cell membrane. Thus, it is likely that the noted increase of Na,K-ATPase activity is due, at least in part, to increased numbers of functional Na,K-ATPases in the basolateral aspect of distal lung cells, although changes in individual enzyme activity cannot be excluded.

Cardiac Output in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– Mice Is Similar to Mice With Intact ßAR Function
Diminished cardiac function due to the absence of ß₁AR and/or ß₂AR in cardiac muscle is a salient concern in the setting of reduced AFC (Figure 5). Accordingly, transthoracic echocardiographic measurements of cardiac output and left ventricular end-diastolic diameter (an index of left ventricular preload and an indirect index of pulmonary hydrostatic pressure) were made in three to four mice per group. Both left ventricular end-diastolic diameter and cardiac output in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice were not significantly different from ß₁AR^+/+/ß₂AR^+/+ mice, limiting concerns that the observed reduction of AFC might be due to unappreciated elevation of left ventricular end diastolic volume and pressure.

View larger version (16K): [in this window] [in a new window]   Figure 5. Cardiac output and left ventricle end-diastolic dimensions, measured with transthoracic echocardiography in ß1AR+/+/ß2AR+/+ and ß1AR–/–/ß2AR–/– mice.

Absence of ß-Receptors Is Associated With Increased Lung Water and Diminished Survival From Acute Lung Injury
To gauge the necessity of alveolar ßARs to lung fluid balance, total lung water content was assessed by measuring wet-to-dry lung weight ratios. Total lung water in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice was not different from ß₁AR^+/+/ß₂AR^+/+ mice (3.931±0.250, 3.93±0.51, and 3.694±0.390, respectively, n=3/group), suggesting that ßAR function may not be required for lung water homeostasis in uninjured mouse lungs. These studies were extended by measuring wet-to-dry weight ratios of lungs of mice with lung injury caused by hyperoxia. Ratios in hyperoxic both ß-receptor knockout strains were 80% greater than hyperoxic wild-type ß₁AR^+/+/ß₂AR^+/+ mice (7.184±0.619 and 7.31±0.77 versus 3.977±0.539, respectively; P<0.002 wild-type versus ß₁AR^–/–/ß₂AR^–/– or ß₁AR^+/+/ß₂AR^–/– mice) (Figure 6A). Histological evaluation of these lungs showed alveolar septal thickening and increased cellularity; however, patchy areas of alveolar edema were noted in lungs from hyperoxic ß₁AR^–/–/ß₂AR^–/– mice (Figure 6B).

View larger version (101K): [in this window] [in a new window]   Figure 6. Effect of ß2AR function on lung water after acute hyperoxic lung injury. A, Lung wet-to-dry ratios from ß1AR+/+/ß2AR+/+, ß1AR+/+/ß2AR–/–, and ß1AR–/–/ß2AR–/– mice exposed to hyperoxia or maintained in room air. *P<0.002 vs room air ß1AR+/+/ß2AR+/+. B, Photomicrographs of hematoxylin and eosin-stained lungs from uninjured and hyperoxic ß1AR+/+/ß2AR+/+ and ß1AR–/–/ß2AR–/– mice exposed to hyperoxia for 66 hours.

To further test the importance of ß₂AR function in this model of lung injury, mice were exposed to hyperoxia for unto 204 hours (Figure 7). The LD₅₀ for the ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice was 72 hours, which was significantly less than ß₁AR^+/+/ß₂AR^+/+ mice (112 hours, P<0.001 ß₁AR^+/+/ß₂AR^–/– or ß₁AR^–/–/ß₂AR^–/– versus ß₁AR^+/+/ß₂AR^+/+). Rescue of ß₂AR function into the alveolar epithelium with adß₂AR resulted in survival of ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– that was the same as adß₂AR infected ß₁AR^+/+/ß₂AR^+/+ controls (LD₅₀: ß₁AR^–/–/ß₂AR^–/–, 192; ß₁AR^+/+/ß₂AR^–/–, 192; ß₁AR^+/+/ß₂AR^+/+, 132 hours; n=6 mice/group, P<0.05 ß₁AR^–/–/ß₂AR^–/–+adß₂AR or ß₁AR^+/+/ß₂AR^–/–+adß₂AR versus ß₁AR^+/+/ß₂AR^+/+). Survival of knockout and wild-type mice transduced with adß₂AR was significantly greater than all other uninfected, adNull-, or sham-infected mice. Infection with adNull did not affect survival in either ß₁AR^+/+/ß₂AR^+/+ or ß₁AR^–/–/ß₂AR^–/– mice (LD₅₀=102 and 84 hours, respectively; P=NS versus same strain uninfected). One adß₂AR-infected ß₁AR^+/+/ß₂AR^+/+ and one adß₂AR ß₁AR^–/–/ß₂AR^–/– mouse survived to the end of the exposure period, which was terminated at 204 hours by agreement with the institutional animal care and use committee. These data strongly suggest that alveolar epithelial ß₂AR function, and not ß₁AR function, is required for adaptation to this lethal lung injury.²⁰

View larger version (18K): [in this window] [in a new window]   Figure 7. Effect of ß2AR function on survival from acute hyperoxic lung injury. Kaplan-Meier plot of survival of ß1AR+/+/ß2AR+/+, ß1AR+/+/ß2AR–/–, and ß1AR–/–/ß2AR–/– mice with and without infection with adß2AR. Survival of adNull-infected ß1AR–/–/ß2AR–/– and ß1AR+/+/ß2AR–/– mice was not different from uninfected mice of the same strain (data not included in graph to improve clarity). Legend includes the LD50 for each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The experiments in this study reveal that mice with no ß₁- or ß₂AR function have significant reductions of the function of key alveolar epithelial transport proteins and severely compromised ability to clear excess alveolar fluid. Specific confirmation of the importance of the ßAR function in the alveolus comes from experiments of ß₂AR rescue into the alveolar epithelium of ß₁AR^–/–/ß₂AR^–/– mice. Doing so improved distal lung ß₂AR function, upregulated Na,K-ATPase activity and amiloride-sensitive Na⁺ entry pathways, and normalized AFC (Figure 3A). Prior studies have shown that both ß₁-²² and ß₂-adrenergic^3,9,23 agonists increase AFC in experimental models. The results of the present studies show similar AFC and survival from hyperoxia in ß₁AR^–/–/ß₂AR^–/– and ß₁AR^+/+/ß₂AR^–/– mice and only a modest response of ß₁AR^+/+/ß₂AR^–/– mice to isoproterenol. Together these findings suggest that ß₂ARs are responsible for the bulk of ß-adrenergic-sensitive AFC in normal mice and that ß₁AR signaling alone is not sufficient for normal rates of clearance of excess alveolar fluid or adaptation to acute lung injury.

Additional evidence for the importance of the ß₂AR to alveolar active Na⁺ transport comes from our studies of ß₁AR^–/–/ß₂AR^–/– mice with acute lung injury. These mice have increased lung water and significantly reduced survival from this model of acute lung injury. These findings might be representative of an inability to cope with the severe stress of an acute lung injury. However, rescue of the ß₂AR only into the alveolar epithelium of ß₁AR^–/–/ß₂AR^–/– or ß₁AR^+/+/ß₂AR^–/– mice conferred the same supranormal survival as in adß₂AR-infected, wild-type mice. Hyperoxia is well suited for these studies as it primarily affects the alveolus. This model and the knockout mice used in this study draw us to the conclusion that epithelial ß₂ARs are required to sustain alveolar function during an acute lung injury that increases total lung water. How receptor gene transfer affects other ßAR sensitive systems (ie, surfactant secretion, antioxidant protein expression) was not tested in these experiments.

A long unanswered question is whether ß-receptors are required to maintain normal alveolar fluid content and AFC rates. This question has been approached in numerous studies in rats,^24,25 rabbits,²⁶ mice,^5,27 dogs,^28,29 sheep,³ guinea pigs,³⁰ and human lung tissue^31,32 via the inclusion of ß-receptor blockers in the alveolar instillate solution only during clearance measurements. Most reported no net effect on unstimulated (ie, no ß-agonists) AFC. A study by our group reported that high doses of propranolol for 3 days reduces AFC by unto 40%¹¹; however, concerns about negative inotropic and chronotropic effects limit applicability of this data to the question of the role of the ßARs in basal AFC. Other groups tested the importance of ß-receptor function to basal AFC with adrenalectomized animals^11,27 or through desensitization of ß-receptors by prolonged infusions of ß-agonists.^33–35 Invariably these studies noted no effect on unstimulated AFC. However, none of these models completely desensitized alveolar ß₂AR function nor were they likely to eliminate cAMP production due to spontaneous receptor activation.³⁶ Similarly, adrenalectomy is not sufficient to totally eliminate serum catecholamines.³⁷ In total, although these studies confirm that ß-receptor activation is an avenue of response to pulmonary edema, they do not confirm or refute a role in regulation of basal AFC. In the present study, we noted that uninjured ß₁AR^–/–/ß₂AR^–/– mice had normal total lung water content (Figure 6A) and that they retain measurable, albeit reduced, active Na⁺ transport (Figure 1A). This data provides new insight that even reduced levels of active transport are sufficient to maintain normal total lung water content and that ß-receptor function may not be required for alveolar fluid homeostasis in uninjured lungs.

Why is AFC reduced in the ß-receptor knockouts? We believe that the findings of decreased amiloride sensitivity and Na,K-ATPase function suggest that ß-receptor signaling is necessary to maintain normal alveolar epithelial Na⁺ transport protein function. We postulate that the low level of active transport noted in ß₁AR^–/–/ß₂AR^–/– mice is due to basal/autonomous function of epithelial transport proteins or is a response of transport proteins (or other receptor signaling systems) to fluid instillation into the alveolus. Prior models indicate that endogenous and exogenous ß-adrenergic agonists enhance the ability of human and animal lung tissue to clear excess alveolar fluid.^25,31,32 The findings of the present study expand this paradigm by demonstrating ß₂ARs are essential components of the pathway by which the alveolus defends itself from acute lung injury and that other epithelial cell receptor and chemo-/osmosensitive regulatory systems are not sufficient to protect from excess alveolar fluid accumulation in the absence of ß₂AR function.

	Acknowledgments

 
This work was supported by the American Heart Association, the Evanston Northwestern Healthcare Research Institute, HL-66211, and HL-71042.

	Footnotes

 
Original received September 29, 2003; resubmission received February 11, 2004; revised resubmission received February 26, 2004; accepted March 2, 2004.

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