Download Upregulation of Alveolar Epithelial Active Na Transport Is

Upregulation of Alveolar Epithelial Active Naⴙ Transport Is
Dependent on ␤2-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
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Abstract—Alveolar epithelial ␤-adrenergic receptor (␤AR) 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 ␤1- or ␤2-adrenergic receptors (␤1AR⫺/⫺/␤2AR⫺/⫺) 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 ␤AR signaling may not be required for
alveolar fluid homeostasis in uninjured lungs. Comparison of isoproterenol-sensitive AFC in mice with ␤1- but not
␤2-adrenergic receptors to ␤1AR⫺/⫺/␤2AR⫺/⫺ mice indicates that the ␤2AR mediates the bulk of ␤-adrenergic–sensitive
alveolar active Na⫹ transport. To test the necessity of ␤AR signaling in acute lung injury, ␤1AR⫺/⫺/␤2AR⫺/⫺,
␤1AR⫹/⫹/␤2AR⫺/⫺, and ␤1AR⫹/⫹/␤2AR⫹/⫹ mice were exposed to 100% oxygen for up to 204 hours. ␤1AR⫺/⫺/␤2AR⫺/⫺
and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice had more lung water and worse survival from this form of acute lung injury than wild-type
controls. Adenoviral-mediated rescue of ␤2-adrenergic receptor (␤2AR) function into the alveolar epithelium of
␤1AR⫺/⫺/␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice normalized distal lung ␤2AR function, alveolar epithelial active Na⫹
transport, and survival from hyperoxia. These findings indicate that ␤AR signaling may not be necessary for basal AFC,
and that ␤2AR is essential for the adaptive physiological response needed to clear excess fluid from the alveolar airspace
of normal and injured lungs. (Circ Res. 2004;94:1091-1100.)
Key Words: alveolar fluid clearance 䡲 pulmonary edema 䡲 ␤2-adrenergic receptor 䡲 adenovirus 䡲 Na⫹ channel
he 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 fluid3–7 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 ␤-adrenergic receptor activation. Stimulation
of alveolar epithelial ␤ARs by endogenous or exogenous
catecholamines accelerates active Na⫹ transport in lung
T
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 ␤AR signaling is essential for
the regulation of alveolar active Na⫹ transport or whether
other mechanisms (eg, intracellular osmo-, redox-, or chemosensitive regulators) can enhance alveolar active transport to
clear pulmonary edema.
The present study was structured to define what contribution alveolar epithelial ␤ARs 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
Original received September 29, 2003; resubmission received February 11, 2004; revised resubmission received February 26, 2004; accepted
March 2, 2004.
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 [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000125623.56442.20
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alveolar epithelial ␤2-adrenergic receptor (␤2AR) function
and that the absence of alveolar ␤2-receptor function compromises survival from an acute lung injury.
Materials and Methods
Adenovirus Propagation and Purification
Replication-incompetent E1a⫺/E3⫺ adenoviruses containing a human
CMV driven human ␤2AR cDNA (ad␤2AR, a gift of Drs Robert
Lefkowitz and Walter Koch, Duke University), an Escherichia coli
lac Z gene (ad␤-gal), or no cDNA (adNull) were propagated,
purified, and titered as previously described.13,14
Animals
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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 ␤2AR
(␤1AR⫹/⫹/␤2AR⫺/⫺), both ␤1AR and ␤2AR genes (␤1AR⫺/⫺/␤2AR⫺/⫺),
and strain-specific ␤1AR⫹/⫹/␤2AR⫹/⫹ (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 ␮L of 100% surfactant (Survanta,
Abbott LaboratoriesL), followed by 200 ␮L of air was administered
via the endotracheal tube.14 A second instillation was performed 5
minutes after the first. All adenovirus-infected animals received
1⫻1011 viral particles 7 days before study. The distribution of gene
transfer using this method was assessed by infecting mice (n⫽4)
with ad␤-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 ␤2AR
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 ␮m) of left lungs fixed with 4% paraformaldehyde were treated with 3% H2O2 before blocking of nonspecific immunoreactivity with nonimmune goat serum. Rabbit antihuman ␤2AR 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 ␤2AR 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
␮g) 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 (Pi Liberation From ATP)
in the Distal Lung
Na,K-ATPase activity was quantified by comparing the amount of
inorganic phosphate (Pi) liberated from ATP over 1 hour by 20 ␮g 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 (Vmax) as
previously described11,18 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
ⴙ
Alveolar Active Na Transport Is Reduced in Mice
With Targeted Deletions of the ␤1AR and/or ␤2AR
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 ␤ARs make to this process, AFC was
measured in mice with targeted deletions of the ␤2AR
(␤1AR⫹/⫹/␤2AR⫺/⫺) and both ␤1AR and ␤2AR (␤1AR⫺/⫺/
␤2AR⫺/⫺) 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 ␮L 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 ␮L. 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 ␤ 1 AR ⫹/⫹ / ␤ 2 AR ⫹/⫹ (wild-type) and
22.2⫾3.0%/30 minutes (n⫽15) in C57BL/6 ␤1AR⫹/⫹/
␤2AR⫹/⫹ control mice (Figure 1A). Importantly, AFC in mice
with ␤1- but no ␤2AR function (␤1AR⫹/⫹/␤2AR⫺/⫺) and mice
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Figure 1. Alveolar fluid clearance in mice. A, Alveolar fluid clearance in uninfected ␤1AR⫹/⫹/␤2AR⫹/⫹ C57BL/6 and wild-type mice and
␤1AR⫹/⫹/␤2AR⫺/⫺ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice in the presence and absence of the ␤2AR-specific agonist procaterol in the alveolar instillate fluid during clearance measurements ( e, untreated; f, procaterol). *P⬍0.05 ␤1AR⫹/⫹/␤2AR⫺/⫺ or ␤1AR⫺/⫺/␤2AR⫺/⫺ 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
ad␤2AR-infected ␤1AR⫹/⫹/␤2AR⫹/⫹, ␤1AR⫹/⫹/␤2AR⫺/⫺, and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice. *P⬍0.05 ad␤2AR-infected vs uninfected, sham-, and
adNull-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice; **P⬍0.05 uninfected, sham-, and adNull-infected ␤1AR⫺/⫺/␤2AR⫺/⫺ mice vs ad␤2AR-infected
␤1AR⫺/⫺/␤2AR⫺/⫺ mice and all groups in ␤1AR⫹/⫹/␤2AR⫹/⫹ mice. C, Effect of inclusion of the ␤2AR specific agonist procaterol in the alveolar instillate of sham- and ad␤2AR-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice ( e, untreated; f, procaterol). *P⬍0.05
procaterol-treated vs untreated, sham-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice; **P⬍0.05 untreated and procaterol-treated ␤1AR⫺/⫺/␤2AR⫺/⫺ mice
vs all other groups. D, Changes in AFC after isoproterenol administration in ␤1AR⫹/⫹/␤2AR⫹/⫹ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice ( 䊐, untreated;
f, isoproterenol). *P⬍0.05 vs untreated ␤1AR⫹/⫹/␤2AR⫹/⫹.
with no ␤1- or ␤2AR function (␤1AR⫺/⫺/␤2AR⫺/⫺) was unto
44% less than in ␤1AR⫹/⫹/␤2AR⫹/⫹ controls (␤1AR⫹/⫹/
␤2AR⫺/⫺, 15.2⫾2.4%/30 minutes, n⫽4; ␤1AR⫺/⫺/␤2AR⫺/⫺,
12.2⫾5.2%/30 minutes, n⫽6; P⬍0.01 ␤1AR⫺/⫺/␤2AR⫺/⫺ or
␤1AR⫹/⫹/␤2AR⫺/⫺ versus wild-type and C57BL/6). In all
experiments, the volume of fluid aspirated from the lungs at
the conclusion of AFC measurements in ␤1AR⫺/⫺/␤2AR⫺/⫺
and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice was greater than from ␤2AR⫹/⫹/
␤1AR⫹/⫹ controls (⬇100 versus ⬇50 ␮L). The inclusion of
the ␤2AR specific agonist procaterol (10⫺8 mol/L) in the
alveolar instillate solution increased clearance in ␤1AR⫹/⫹/
␤2AR⫹/⫹ 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 ␤1AR⫺/⫺/␤2AR⫺/⫺ mice (11.6⫾3.1%
per 30 minutes, n⫽4) or ␤1AR⫹/⫹/␤2AR⫺/⫺ mice (15.0⫾1.5%
per 30 minutes, n⫽4) (Figure 1B). The reduced basal AFC
rates in the ␤1AR⫺/⫺/␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice
confirm an important role for ␤2AR signaling in the regulation of alveolar epithelial active Na⫹ transport in mice.
␤2AR Gene Transfer Normalizes Alveolar Active
Naⴙ Transport in ␤1ARⴚ/ⴚ/␤2ARⴚ/ⴚ and
␤1ARⴙ/ⴙ/␤2ARⴚ/ⴚ Mice
To assess the contribution of ␤AR function to active Na⫹
transport in the alveolar epithelium, ␤1AR⫺/⫺/␤2AR⫺/⫺,
␤1AR⫹/⫹/␤2AR⫺/⫺, and ␤2AR⫹/⫹/␤1AR⫹/⫹ mice were infected
with a replication-incompetent, E1a⫺/E3⫺ recombinant adenovirus that expresses a human ␤2AR (ad␤2AR) 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 ad␤-gal indicated that 68⫾5%
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Figure 2. Transgene expression in mouse lungs. A, Representative lungs from a ␤1AR⫹/⫹/␤2AR⫹/⫹ (wild-type) mouse infected with
1⫻1011 viral particles of ad␤-gal and stained with X-gal 7 days later. Adjacent photomicrographs are of 3-␮m sections of paraffinimbedded lungs from ␤1AR⫹/⫹/␤2AR⫹/⫹ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice (original magnification 40⫻). Transfection efficiency was enumerated
in 3 mice/group as the number of alveoli with at least 1 cell with perinuclear ␤-galactosidase activity. Transfection efficiency was
70⫾12% in ␤1AR⫹/⫹/␤2AR⫹/⫹ mice and 68⫾14% in ␤1AR⫺/⫺/␤2AR⫺/⫺ mice (P⫽NS). B, Representative Western blot of whole cell membrane fractions from peripheral lungs of ␤1AR⫹/⫹/␤2AR⫹/⫹ mice infected with vehicle (sham), adNull, or ad␤2AR showing the presence of
a human ␤2AR only in lungs infected with ad␤2AR. C, Immunostaining of whole lungs using an anti-human ␤2AR antibody showing the
presence of a human ␤2AR only in the alveoli of ad␤2AR-infected animals. Circumferential pattern of immunostaining is consistent with
transduction of both alveolar type 1 and type 2 epithelial cells. Original magnification: 400⫻.
and 70⫾7% of alveoli in ␤1AR⫹/⫹/␤2AR⫹/⫹ and ␤1AR⫺/⫺/
␤2AR⫺/⫺ mice, respectively, had evidence of transgene expression (Figure 2A). Immunostaining of lungs of ad␤2AR
infected mice for human ␤2AR 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 ␤2AR function into ␤1AR⫺/⫺/
␤2AR⫺/⫺ mice with ad␤2AR 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 ␤1AR⫹/⫹/
␤2AR⫺/⫺ mice (n⫽4). These rates of AFC were not different
from uninfected ␤1AR⫹/⫹/␤2AR⫹/⫹ C57BL/6 or wild-type
mice, although the magnitude of increase in ␤1AR⫹/⫹/
␤2AR⫺/⫺ was slightly greater than ␤1AR⫺/⫺/␤2AR⫺/⫺
(P⫽0.045). ␤2AR gene transfer also increased clearance in
strain-specific wild-type (␤1AR⫹/⫹/␤2AR⫹/⫹) mice (44.7% to
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31.7.1⫾4.4%/30 minutes, n⫽4) (Figure 1B). Sham and
adNull infection did not affect AFC in the ␤1AR⫺/⫺/␤2AR⫺/⫺,
␤1AR⫹/⫹/␤2AR⫺/⫺, or ␤1AR⫹/⫹/␤2AR⫹/⫹ groups. Thus, ␤2AR
function in ⬇70% of alveoli is sufficient to normalize AFC in
␤1AR⫺/⫺/␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice. These experiments are the first to both localize transgene expression and
demonstrate a relevant physiological effect after adenoviralmediated gene transfer in the distal lung of mice.
Inclusion of the specific ␤2-agonist procaterol in the
isotonic alveolar instillate fluid had no effect on AFC in
␤1AR⫹/⫹/␤2AR⫹/⫹ or ␤1AR⫺/⫺/␤2AR⫺/⫺ mice after infection
with ad␤2AR (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 ␤2ARsensitive AFC without the addition of exogenous catecholamines in rats, and that the human ␤2AR cDNA used in this
study is not constitutively active.11 Prior studies in adrenalectomized rodents suggests that increased ␤AR function
after ␤2AR gene transfer is due to increased numbers of
receptors in the cell membrane and possibly enhanced sensitivity to endogenous catecholamines.
␤1AR Signaling Does Not Contribute to Alveolar
Active Naⴙ Transport to the Same Degree
as ␤2ARs
The contribution of ␤1AR signaling to basal AFC was
measured by including the nonspecific ␤-agonist isoproterenol (10⫺4 mol/L) in the alveolar instillate of ␤1AR⫹/⫹/␤2AR⫺/⫺
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 ␤1AR⫹/⫹/␤2AR⫹/⫹ controls (21.9⫾4.0% per
30 minutes). Isoproterenol increased clearance by 41% (to
30.9⫾2.3% per 30 minutes) in strain-specific ␤1AR⫹/⫹/
␤2AR⫹/⫹ mice (P⫽0.001 isoproterenol treated ␤1AR⫹/⫹/
␤2AR⫺/⫺ versus isoproterenol-treated ␤1AR⫹/⫹/␤2AR⫹/⫹). The
greater degree of change in these mice is probably due to
isoproterenol-mediated activation of ␤2ARs. Importantly,
basal clearance in untreated ␤1AR⫹/⫹/␤2AR⫺/⫺ was not statistically different from that of untreated ␤1AR⫺/⫺/␤2AR⫺/⫺ mice
(P⫽0.35) (Figure 1A). These data provide evidence that
␤1ARs do not contribute to basal AFC to the same degree as
␤2ARs in normal mice. The use of ␤AR knockout mice in
these studies is particularly relevant as the absence of the
␤2AR might allow for compensatory expansion of the role of
the ␤1AR in regulating AFC in ␤1AR⫹/⫹/␤2AR⫺/⫺ mice, hence
the modest changes in AFC in isoproterenol treated ␤1AR⫹/⫹/
␤2AR⫺/⫺ mice may overstate the contribution of the ␤1AR to
AFC in wild-type mice.
␤2AR Rescue Normalizes Peripheral Lung
␤-Receptor Function in ␤1ARⴚ/ⴚ/␤2ARⴚ/ⴚ Mice
Baseline cAMP production by whole-cell membranes from
the peripheral lungs of sham-infected ␤1AR⫺/⫺/␤2AR⫺/⫺ 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 ␤2AR agonist procaterol (10⫺8 mol/L⫻30
minutes) (Figure 3A). Infection of ␤1AR⫺/⫺/␤2AR⫺/⫺ and
Figure 3. A, ␤-receptor function in peripheral lung membranes.
Baseline (untreated), procaterol-responsive, and forskolininduced cAMP production by whole-cell membrane fractions
isolated from peripheral lung tissue (䡺, untreated; 䡵, procaterol;
, forskolin). *P⬍0.05 vs same treatment group ␤1AR⫹/⫹/␤2AR⫹/⫹
mice. B, Cyclic-AMP content in peripheral lung tissue
homogenates.
␤1AR⫹/⫹/␤2AR⫹/⫹ mice with ad␤2AR had no significant effect
on basal cAMP production.
Procaterol-induced cAMP production (an index of ␤2AR
function) by cell membranes from ad␤2AR-infected ␤1AR⫺/⫺/
␤2AR⫺/⫺ mice was 5.73⫾0.4 pmol/mg protein, which is
similar to that in sham-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice
(3.8⫾0.4 pmol/mg protein) but significantly less than
ad␤2AR-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice (10.7⫾1.4 pmol/mg
protein). Thus, ␤2AR gene transfer rescues normal ␤2AR
function in the distal lung of ␤1AR⫺/⫺/␤2AR⫺/⫺ mice and
results in increased receptor function in wild-types. A small
increase in procaterol-sensitive cAMP production by membranes from adNull-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ 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
␤1AR⫺/⫺/␤2AR⫺/⫺ mice was not different from strain-specific
␤1AR⫹/⫹/␤2AR⫹/⫹ mice (Figure 3B). Thus, low intracellular
cAMP is not the explanation for the reduced active Na⫹
transport noted in the ␤1AR⫺/⫺/␤2AR⫺/⫺ mice.
Forskolin-induced cAMP production, an index of adenylyl
cyclase function, was lower in all ␤1AR⫺/⫺/␤2AR⫺/⫺ groups
(Figure 3A). ␤2AR gene transfer increased forskolin-induced
cAMP production in both wild-type and knockout mice
infected with ad␤2AR. These findings suggest that distal lung
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Figure 4. A, Effect of amiloride on AFC. Data are percent reduction AFC (as compared with untreated controls) in ␤1AR⫹/⫹/
␤2AR⫹/⫹ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice. *P⬍0.05 ad␤2AR-infected vs
sham- and adNull-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice; **P⬍0.05
sham- and adNull-infected ␤1AR⫺/⫺/␤2AR⫺/⫺ 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 ␤1AR⫹/⫹/␤2AR⫹/⫹ mice.
␤ARs may participate in the regulation of their downstream
signaling pathways or that the absence of basal/tonic signaling influences of the ␤2AR on adenylyl cyclase could result in
its downregulation. Nevertheless, membranes from ␤1AR⫺/⫺/
␤2AR⫺/⫺ mice retained responsiveness to forskolin, indicating
that signaling systems downstream from the ␤2AR are
preserved.
␤-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 ␤1AR⫺/⫺/
␤2AR⫺/⫺ 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 ␤1AR⫺/⫺/␤2AR⫺/⫺ mice, respectively,
which was significantly less than the reduction noted in sham
and adNull-infected ␤1AR⫹/⫹/␤2AR⫹/⫹ mice (50 and 45%,
respectively; P⬍0.05 sham or adNull infected ␤1AR⫺/⫺/
␤2AR⫺/⫺ versus sham or adNull-infected ␤1AR⫹/⫹/␤2AR⫹/⫹)
and is suggestive of diminished amiloride-sensitive Na⫹
transporter function. Rescue of ␤2AR function into the
alveolar epithelium of ␤1AR⫺/⫺/␤2AR⫺/⫺ mice restored
amiloride-sensitivity to nearly normal (44% reduction of
AFC; P⫽0.45 versus ␤1AR⫹/⫹/␤2AR⫹/⫹). Amiloride decreased AFC to a greater degree in ad␤2AR-infected, ␤1AR⫹/⫹/
␤2AR⫹/⫹ animals (59% to 13.0⫾3.7%/30 minutes, n⫽4) than
in shams, indicating that ␤2AR 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 ␤AR signaling.
The impact of ␤AR signaling on Na,K-ATPase function
was assessed by measuring Na,K-ATPase activity (ouabainsensitive liberation of Pi from ATP) by basolateral membranes isolated from the peripheral lung. Na,K-ATPase activity in sham and adNull-infected mice ␤1AR⫺/⫺/␤2AR⫺/⫺
was ⬇30% of that in similarly infected ␤1AR⫹/⫹/␤2AR⫹/⫹
mice (P⬍0.02 sham or adNull ␤1AR⫺/⫺/␤2AR⫺/⫺ mice versus
sham or adNull ␤1AR⫹/⫹/␤2AR⫹/⫹ mice) (Figure 4B). Restoration of ␤AR function in the alveolar epithelium with
ad␤2AR increased maximal Na,K-ATPase activity more than
20-fold to a level similar to ad␤2AR-infected ␤1AR⫹/⫹/
␤2AR⫹/⫹ 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 ␤1ARⴚ/ⴚ/␤2ARⴚ/ⴚ and
␤1ARⴙ/ⴙ/␤2ARⴚ/ⴚ Mice Is Similar to Mice With
Intact ␤AR Function
Diminished cardiac function due to the absence of ␤1AR
and/or ␤2AR 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
␤1AR⫺/⫺/␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice were not significantly different from ␤1AR⫹/⫹/␤2AR⫹/⫹ mice, limiting
concerns that the observed reduction of AFC might be due to
unappreciated elevation of left ventricular end diastolic volume and pressure.
Absence of ␤-Receptors Is Associated With
Increased Lung Water and Diminished Survival
From Acute Lung Injury
To gauge the necessity of alveolar ␤ARs to lung fluid
balance, total lung water content was assessed by measuring
Mutlu et al
Role of ␤-Receptors in Alveolar Active Transport
1097
knockout and wild-type mice transduced with ad␤2AR was
significantly greater than all other uninfected, adNull-, or
sham-infected mice. Infection with adNull did not affect
survival in either ␤1AR⫹/⫹/␤2AR⫹/⫹ or ␤1AR⫺/⫺/␤2AR⫺/⫺ mice
(LD50⫽102 and 84 hours, respectively; P⫽NS versus same
strain uninfected). One ad␤2AR-infected ␤1AR⫹/⫹/␤2AR⫹/⫹
and one ad␤2AR ␤1AR⫺/⫺/␤2AR⫺/⫺ 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 ␤2AR
function, and not ␤1AR function, is required for adaptation to
this lethal lung injury.20
Discussion
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Figure 5. Cardiac output and left ventricle end-diastolic dimensions, measured with transthoracic echocardiography in ␤1AR⫹/⫹/
␤2AR⫹/⫹ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice.
wet-to-dry lung weight ratios. Total lung water in ␤1AR⫺/⫺/
␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice was not different from
␤1AR⫹/⫹/␤2AR⫹/⫹ mice (3.931⫾0.250, 3.93⫾0.51, and
3.694⫾0.390, respectively, n⫽3/group), suggesting that ␤AR
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
␤-receptor knockout strains were ⬇80% greater than hyperoxic wild-type ␤1AR⫹/⫹/␤2AR⫹/⫹ mice (7.184⫾0.619 and
7.31⫾0.77 versus 3.977⫾0.539, respectively; P⬍0.002 wildtype versus ␤1AR⫺/⫺/␤2AR⫺/⫺ or ␤1AR⫹/⫹/␤2AR⫺/⫺ 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 ␤1AR⫺/⫺/␤2AR⫺/⫺ mice (Figure 6B).
To further test the importance of ␤2AR function in this
model of lung injury, mice were exposed to hyperoxia for
unto 204 hours (Figure 7). The LD50 for the ␤1AR⫺/⫺/␤2AR⫺/⫺
and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice was 72 hours, which was significantly less than ␤1AR⫹/⫹/␤2AR⫹/⫹ mice (112 hours, P⬍0.001
␤1AR⫹/⫹/␤2AR⫺/⫺ or ␤1AR⫺/⫺/␤2AR⫺/⫺ versus ␤1AR⫹/⫹/
␤2AR⫹/⫹). Rescue of ␤2AR function into the alveolar epithelium with ad␤2AR resulted in survival of ␤1AR⫺/⫺/␤2AR⫺/⫺
and ␤1AR⫹/⫹/␤2AR⫺/⫺ that was the same as ad␤2AR infected
␤1AR⫹/⫹/␤2AR⫹/⫹ controls (LD50: ␤1AR⫺/⫺/␤2AR⫺/⫺, 192;
␤1AR⫹/⫹/␤2AR⫺/⫺, 192; ␤1AR⫹/⫹/␤2AR⫹/⫹, 132 hours; n⫽6
mice/group, P⬍0.05 ␤1AR⫺/⫺/␤2AR⫺/⫺⫹ad␤2AR or ␤1AR⫹/⫹/
␤2AR⫺/⫺⫹ad␤2AR versus ␤1AR⫹/⫹/␤2AR⫹/⫹). Survival of
The experiments in this study reveal that mice with no ␤1- or
␤2AR 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 ␤AR function in the
alveolus comes from experiments of ␤2AR rescue into the
alveolar epithelium of ␤1AR⫺/⫺/␤2AR⫺/⫺ mice. Doing so
improved distal lung ␤2AR function, upregulated Na,KATPase activity and amiloride-sensitive Na⫹ entry pathways,
and normalized AFC (Figure 3A). Prior studies have shown
that both ␤1-22 and ␤2-adrenergic3,9,23 agonists increase AFC
in experimental models. The results of the present studies
show similar AFC and survival from hyperoxia in ␤1AR⫺/⫺/
␤2AR⫺/⫺ and ␤1AR⫹/⫹/␤2AR⫺/⫺ mice and only a modest
response of ␤1AR⫹/⫹/␤2AR⫺/⫺ mice to isoproterenol. Together
these findings suggest that ␤2ARs are responsible for the bulk
of ␤-adrenergic–sensitive AFC in normal mice and that ␤1AR
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 ␤2AR to
alveolar active Na⫹ transport comes from our studies of
␤1AR⫺/⫺/␤2AR⫺/⫺ 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 ␤2AR only into
the alveolar epithelium of ␤1AR⫺/⫺/␤2AR⫺/⫺ or ␤1AR⫹/⫹/
␤2AR⫺/⫺ mice conferred the same supranormal survival as in
ad␤2AR-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 ␤2ARs are required to sustain
alveolar function during an acute lung injury that increases
total lung water. How receptor gene transfer affects other
␤AR sensitive systems (ie, surfactant secretion, antioxidant
protein expression) was not tested in these experiments.
A long unanswered question is whether ␤-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 tissue31,32 via the inclusion of ␤-receptor
blockers in the alveolar instillate solution only during clearance measurements. Most reported no net effect on unstimulated (ie, no ␤-agonists) AFC. A study by our group reported
that high doses of propranolol for 3 days reduces AFC by
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Figure 6. Effect of ␤2AR function on lung water after acute hyperoxic lung injury. A, Lung wet-to-dry ratios from ␤1AR⫹/⫹/␤2AR⫹/⫹,
␤1AR⫹/⫹/␤2AR⫺/⫺, and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice exposed to hyperoxia or maintained in room air. *P⬍0.002 vs room air ␤1AR⫹/⫹/␤2AR⫹/⫹. B,
Photomicrographs of hematoxylin and eosin–stained lungs from uninjured and hyperoxic ␤1AR⫹/⫹/␤2AR⫹/⫹ and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice
exposed to hyperoxia for 66 hours.
unto 40%11; however, concerns about negative inotropic and
chronotropic effects limit applicability of this data to the
question of the role of the ␤ARs in basal AFC. Other groups
tested the importance of ␤-receptor function to basal AFC
with adrenalectomized animals11,27 or through desensitization
of ␤-receptors by prolonged infusions of ␤-agonists.33–35
Invariably these studies noted no effect on unstimulated AFC.
However, none of these models completely desensitized
alveolar ␤2AR 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 ␤-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 ␤1AR⫺/⫺/␤2AR⫺/⫺ 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
␤-receptor function may not be required for alveolar fluid
homeostasis in uninjured lungs.
Why is AFC reduced in the ␤-receptor knockouts? We
believe that the findings of decreased amiloride sensitivity
and Na,K-ATPase function suggest that ␤-receptor signaling
is necessary to maintain normal alveolar epithelial Na⫹
transport protein function. We postulate that the low level of
active transport noted in ␤1AR⫺/⫺/␤2AR⫺/⫺ mice is due to
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Role of ␤-Receptors in Alveolar Active Transport
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Figure 7. Effect of ␤2AR function on survival from acute hyperoxic lung injury. Kaplan-Meier plot of survival of ␤1AR⫹/⫹/␤2AR⫹/⫹, ␤1AR⫹/⫹/
␤2AR⫺/⫺, and ␤1AR⫺/⫺/␤2AR⫺/⫺ mice with and without infection with ad␤2AR. Survival of adNull-infected ␤1AR⫺/⫺/␤2AR⫺/⫺ and ␤1AR⫹/⫹/
␤2AR⫺/⫺ 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.
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 ␤-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 ␤2ARs 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 ␤2AR function.
Acknowledgments
This work was supported by the American Heart Association, the
Evanston Northwestern Healthcare Research Institute, HL-66211,
and HL-71042.
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Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
Upregulation of Alveolar Epithelial Active Na+ Transport Is Dependent on β2-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 and Phillip Factor
Circ Res. 2004;94:1091-1100; originally published online March 11, 2004;
doi: 10.1161/01.RES.0000125623.56442.20
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
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http://circres.ahajournals.org/content/suppl/2004/05/12/94.8.1091.DC1
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Adenovirus propagation and purification. Replication-incompetent E1a-/E3human type 5 adenoviruses containing a human CMV driven human β2AR cDNA
(adβ2AR, a gift of Drs. Robert Lefkowitz and Walter Koch, Duke University), an E. coli
lac Z gene (adβ-gal), or no cDNA (adNull) were propagated, purified and titered on
HEK293 cells as previously described
1,2
.
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, IN). Mice
with targeted deletions of the β2AR (β1AR+/+/β2AR-/-), both β1AR and β2AR genes
(β1AR-/-/β2AR-/-), and strain specific β1AR+/+/β2AR+/+ (wild-type) mice, were from Dr.
Brian Kobilka (Stanford University)
3,4
.
Adenovirus delivery to mouse lungs. Mice were anesthetized with pentobarbital
(75mg/kg, i.p.) and orally intubated under direct visualization using a 2 cm, 20g
plastic intravenous catheter. Adenovirus, in 25µl of 100% surfactant (Survanta,
Abbott Laboratories, Chicago, IL), followed by 200 µl of air was administered via the
endotracheal tube using methods adapted from prior rat experiments 5. A second
instillation was performed 5 minutes following the first. All adenovirus infected
animals received 1x1011 viral particles 7 days prior to study. The distribution of gene
transfer using this method was assessed by infecting mice (n=4) with adβ-gal as
previously described 5.
Alveolar fluid clearance measurement. The method used in this study to quantify
alveolar fluid clearance was from Hardiman
6
except that mice were maintained
supine during the 30 minute period of measurement. Briefly, mice were anesthetized
with diazepam (5mg/kg, i.p.) and pentobarbital (50mg/kg, i.p. given 10 minutes
after diazepam). The trachea was then exposed and cannulated with a 1cm, 20gauge plastic intravenous catheter (Angiocath, Becton-Dickenson, Sandy, UT). Mice
were paralyzed with pancuronium bromide (0.04 mg, i.p. Gensia Pharmaceuticals,
Irvine, CA) and ventilated with a tidal volume of 10 ml/kg at a frequency of 160
breaths per minute using 100% oxygen delivered via a small animal ventilator
(Harvard Apparatus, Holliston, MA). Body temperature was maintained at 37°C using
a heating pad (Gaymar Industries Inc., Orchard Park, NY). Heart rate and rhythm
were monitored continuously (LifePak 6, Physio-Control, Redmond, WA).
Following a 10 minute equilibrium period 300µl of an isosmolar (320mOsm) 7,
0.9% NaCl solution containing 5% acid-free bovine serum albumin (BSA, Sigma, St.
Louis, MO) was instilled into the endotracheal catheter over 10 seconds followed by
100µl of air to position the fluid in the alveolar space
6
. The animals were kept
supine, inclined to 30-degrees. Mechanical ventilation was continued for 30 minutes
whereupon the chest was opened to produce bilateral pneumothoraces to facilitate
aspiration of remaining the alveolar fluid via the tracheal catheter. Protein
concentration was measured using a modified Bradford assay (Bio-Rad laboratories,
Hercules, CA).
Alveolar fluid clearance was calculated using the following equation:
AFC= 1-(C0/C30)
Where C0, is the protein concentration of the instillate before instillation and C30, is
the protein concentration of the sample obtained at the end of 30 minutes of
mechanical ventilation 6. Clearance is expressed as a percentage of total instilled
volume (%/30min). In some experiments procaterol (a specific β2AR agonist, 10-8 M)
or amiloride (10-3 M) were administered in the instillate. Amiloride sensitivity is
reported as % reduction AFC as compared to similarly treated mice not exposed to
amiloride.
Immunohistochemistry. Longitudinal sections (3µm) of left lungs fixed with 4%
paraformaldehyde were treated with 3% H2O2 prior to blocking of non-specific
immunoreactivity with non-immune goat serum. Rabbit anti-human β2AR antibody
(1:500 dilution, Santa Cruz Scientific, Santa Cruz, CA) was added for 1 hour at room
temperature prior to washing and immunodetection using a fluorescene-linked
secondary antibody (Vector Elite ABC kit, Vector Laboratories, New Castle-uponTyne, UK).
Whole and basolateral cell membrane isolation and western analysis.
Membrane proteins were obtained by homogenizing lung tissue collected from the
peripheral 1-2 mm of each lobe (peripheral lung, ~ 500mg wet weight) as previously
described
8,9
. For western analysis 10 µg of whole cell membrane protein was
separated by 10% SDS-PAGE, electrophoretically transferred to nitrocellulose and
probed with a rabbit anti-human β2AR antibody (Santa Cruz Scientific, Santa Cruz,
CA). An immunperoxidase based chemiluminescent detection system (ECL+ Plus,
Amersham Corporation, Arlington Heights, IL) was used for immunodetection.
Basolateral cell membranes were isolated as previously described
8,9
.
Measurement of cAMP levels. Cyclic-AMP production by whole cell membrane
fractions from peripheral lung tissue over 30 minutes was measured using 5-10µg of
membrane protein using a radioimmunoassay (Amprep SAX, NEN/Perkin Elmer,
Boston, MA) as described previously
10
. Measurements were performed in triplicate
and are presented as pmol cAMP/mg membrane protein.
Na,K-ATPase function (Pi liberation from ATP) in the distal lung. Na,K-ATPase
activity was quantified by comparing the amount of inorganic phosphate (Pi)
liberated from ATP over 1 hour by 20 µg of basolateral cell membrane protein
isolated from the peripheral lung as previously described
8,9
. Comparison of Pi
liberation in the presence and absence of the Na,K-ATPase inhibitor ouabain is used
to quantify Na,K-ATPase activity. Conditions used maximize Na,K-ATPase activity
(Vmax) to produce an index of functional, membrane-bound receptor number.
Results are expressed as mean nmol of Pi/mg of protein/hour of triplicate
measurements from three mice/group.
Echocardiographic assessment of cardiac function. Light anesthesia was
induced with halothane for 5-10 minutes until the heart rate stabilized to within 10%
of initial rate in 3 animals/group. Echocardiography was performed using a phase
array 12 MHz and linear 6-15MHz transducers and an Agilent 5500 echocardiography
machine. To minimize the effects of anesthesia, images were obtained as animals
began to demonstrate spontaneous movement. Parasternal long and short axis Mmode images were used to obtain average left ventricular end-diastolic (LVEDd)
dimensions of three consecutive heart cycles. Aortic outflow tract diameter was
determined in the parasternal long axis by M-mode, and 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
11
.
Induction of Acute Lung Injury. Wild-type and β2AR-/-/β1AR-/- mice (n=3-4
mice/group) were exposed to hyperoxia (100% normobaric O2) in 2 sets of
experiments. To determine total lung water content (an index of injury) mice were
exposed to hyperoxia for 66 hours prior to anticoagulation (heparin, 1000U, i.p.),
anesthesia, exsanguination and collection of tissue from the distal left lung for
measurement of wet/dry ratios using a heated vacuum centrifuge as previously
described
12
. The right lungs of these mice were fixed in paraformaldehyde and
imbedded in paraffin for histologic evaluation
13,14
. Survival studies were conducted
by exposing mice to hyperoxia for up to 204 hours. This duration of exposure was
established by agreement with the Evanston Northwestern Healthcare IACUC.
Surviving animals were enumerated at 12 hour intervals.
Data Analysis. All values are reported as means ± standard deviation. Statistical
comparison among groups was performed using one-way ANOVA (GraphPad Prism,
GraphPad Software, Inc., San Diego, CA). Comparison of survival among groups
exposed to hyperoxia was performed with using the Kaplan-Meier method to
determine the LD50 (Graphpad Prism). Statistical significance in all experiments was
defined as P<0.05.
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