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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 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 1091 1092 Circulation Research April 30, 2004 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 (ad2AR, 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 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 Mutlu et al Role of -Receptors in Alveolar Active Transport 1093 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR-infected 1AR⫹/⫹/2AR⫹/⫹, 1AR⫹/⫹/2AR⫺/⫺, and 1AR⫺/⫺/2AR⫺/⫺ mice. *P⬍0.05 ad2AR-infected vs uninfected, sham-, and adNull-infected 1AR⫹/⫹/2AR⫹/⫹ mice; **P⬍0.05 uninfected, sham-, and adNull-infected 1AR⫺/⫺/2AR⫺/⫺ mice vs ad2AR-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 ad2AR-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 (ad2AR) 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% 1094 Circulation Research April 30, 2004 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR showing the presence of a human 2AR only in lungs infected with ad2AR. C, Immunostaining of whole lungs using an anti-human 2AR antibody showing the presence of a human 2AR only in the alveoli of ad2AR-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 ad2AR 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 ad2AR 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 Mutlu et al Role of -Receptors in Alveolar Active Transport 1095 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR (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 ad2AR had no significant effect on basal cAMP production. Procaterol-induced cAMP production (an index of 2AR function) by cell membranes from ad2AR-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 ad2AR-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 ad2AR. These findings suggest that distal lung 1096 Circulation Research April 30, 2004 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR-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 ad2AR-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 ad2AR increased maximal Na,K-ATPase activity more than 20-fold to a level similar to ad2AR-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 ad2AR 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 ad2AR-infected 1AR⫹/⫹/2AR⫹/⫹ and one ad2AR 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 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR resulted in survival of 1AR⫺/⫺/2AR⫺/⫺ and 1AR⫹/⫹/2AR⫺/⫺ that was the same as ad2AR infected 1AR⫹/⫹/2AR⫹/⫹ controls (LD50: 1AR⫺/⫺/2AR⫺/⫺, 192; 1AR⫹/⫹/2AR⫺/⫺, 192; 1AR⫹/⫹/2AR⫹/⫹, 132 hours; n⫽6 mice/group, P⬍0.05 1AR⫺/⫺/2AR⫺/⫺⫹ad2AR or 1AR⫹/⫹/ 2AR⫺/⫺⫹ad2AR 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 ad2AR-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 1098 Circulation Research April 30, 2004 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 Mutlu et al Role of -Receptors in Alveolar Active Transport 1099 Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 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 ad2AR. 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. References 1. Matthay MA, Folkesson HG, Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev. 2002;82:569 – 600. 2. Factor P. Role and regulation of lung Na,K-ATPase. Cell Mol Biol. 2001;47:347–361. 3. Berthiaume Y, Staub NC, Matthay MA. -Adrenergic agonists increase lung liquid clearance in anesthetized sheep. J Clin Invest. 1987;79: 335–343. 4. Azzam ZS, Saldias FJ, Comellas A, Ridge KM, Rutschman DH, Sznajder JI. Catecholamines increase lung edema clearance in rats with increased left atrial pressure. J Appl Physiol. 2001;90:1088 –1094. 5. Icard P, Saumon G. Alveolar sodium and liquid transport in mice. Am J Physiol. 1999;277:L1232–L1238. 6. Jayr C, Garat C, Meignan M, Pittet JF, Zelter M, Matthay MA. Alveolar liquid and protein clearance in anesthetized ventilated rats. J Appl Physiol. 1994;76:2636 –2642. 7. Kim KJ, Cheek JM, Crandall ED. Contribution of active Na⫹ and Cl⫺ fluxes to net ion transport by alveolar epithelium. Respir Physiol. 1991; 85:245–256. 8. Factor P, Dumasius V, Saldias F, Brown LA, Sznajder JI. Adenovirusmediated transfer of an Na⫹/K⫹-ATPase 1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats. Hum Gene Ther. 2000;11:2231–2242. 9. Lasnier JM, Wangensteen OD, Schmitz LS, Gross CR, Ingbar DH. Terbutaline stimulates alveolar fluid resorption in hyperoxic lung injury. J Appl Physiol. 1996;81:1723–1729. 10. Maron MB. Dose-response relationship between plasma epinephrine concentration and alveolar liquid clearance in dogs. J Appl Physiol. 1998; 85:1702–1707. 11. Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, Factor P. 2-Adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats. Circ Res. 2001;89:907–914. 12. Yue G, Shoemaker R, Matalon S. Regulation of low-amiloride-affinity sodium channels in alveolar type II cells. Am J Physiol. 1994;267: L94 –L100. 13. Factor P, Senne C, Dumasius V, Ridge K, Ari Jaffe H, Uhal B, Gao Z, Sznajder J. Overexpression of the Na,K-ATPase ␣1 subunit increases Na,K-ATPase function in A549 cells. Am J Respir Cell Mol Biol. 1998; 18:741–749. 14. Factor P, Saldias F, Ridge K, Dumasius V, Zabner J, Jaffe HA, Blanco G, Barnard M, Mercer R, Perrin R, Sznajder JI. Augmentation of lung liquid clearance via adenoviral-mediated gene transfer of the Na,K-ATPase 1 subunit. J Clin Invest. 1998;102:1142–1150. 15. Chruscinski AJ, Rohrer DK, Schauble E, Desai KH, Bernstein D, Kobilka BK. Targeted disruption of the 2 adrenergic receptor gene. J Biol Chem. 1999;274:16694 –16700. 16. Rohrer D, Chruscinski A, Schauble E, Bernstein D, Kobilka B. Cardiovascular and metabolic alterations in mice lacking both 1 and 2-adrenergic receptors. J Biol Chem. 1999;274:16701–16708. 17. Hardiman KM, Lindsey JR, Matalon S. Lack of amiloride-sensitive transport across alveolar and respiratory epithelium of iNOS(⫺/⫺) mice in vivo. Am J Physiol Lung Cell Mol Physiol. 2001;281:L722–L731. 18. Azzam ZS, Dumasius V, Saldias FJ, Adir Y, Sznajder JI, Factor P. Na,K-ATPase overexpression improves alveolar fluid clearance in a rat model of elevated left atrial pressure. Circulation. 2002;105:497–501. 19. Hollenberg SM, Dumasius A, Easington C, Colilla SA, Neumann A, Parrillo JE. Characterization of a hyperdynamic murine model of resuscitated sepsis using echocardiography. Am J Respir Crit Care Med. 2001;164:891– 895. 20. Factor P, Ridge K, Alverdy J, Sznajder J. Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen. J Appl Physiol. 2000;89:1759 –1765. 21. Minakata Y, Suzuki S, Grygorczyk C, Dagenais A, Berthiaume Y. Impact of -adrenergic agonist on Na⫹ channel and Na⫹-K⫹-ATPase expression in alveolar type II cells. Am J Physiol. 1998;275:L414 –L422. 22. Sakuma T, Tuchihara C, Ishigaki M, Osanai K, Nambu Y, Toga H, Takahashi K, Ohya N, Kurihara T, Matthay MA. Denopamine, a 1100 23. 24. 25. 26. 27. 28. 29. Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017 30. Circulation Research April 30, 2004 1-adrenergic agonist, increases alveolar fluid clearance in ex vivo rat and guinea pig lungs. J Appl Physiol. 2001;90:10 –16. Crandall ED, Heming TA, Palombo RL, Goodman BE. Effects of terbutaline on sodium transport in isolated perfused rat lung. J Appl Physiol. 1986;60:289 –294. Borjesson A, Norlin A, Wang X, Andersson R, Folkesson HG. TNF-␣ stimulates alveolar liquid clearance during intestinal ischemia-reperfusion in rats. Am J Physiol Lung Cell Mol Physiol. 2000;278:L3–L12. Pittet JF, Wiener-Kronish JP, McElroy MC, Folkesson HG, Matthay MA. Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats. J Clin Invest. 1994; 94:663– 671. Smedira N, Gates L, Hastings R, Jayr C, Sakuma T, Pittet JF, Matthay MA. Alveolar and lung liquid clearance in anesthetized rabbits. J Appl Physiol. 1991;70:1827–1835. Fukuda N, Folkesson HG, Matthay MA. Relationship of interstitial fluid volume to alveolar fluid clearance in mice: ventilated vs. in situ studies. J Appl Physiol. 2000;89:672– 679. Berthiaume Y, Broaddus VC, Gropper MA, Tanita T, Matthay MA. Alveolar liquid and protein clearance from normal dog lungs. J Appl Physiol. 1988;65:585–593. Lane SM, Maender KC, Awender NE, Maron MB. Adrenal epinephrine increases alveolar liquid clearance in a canine model of neurogenic pulmonary edema. Am J Respir Crit Care Med. 1998;158:760 –768. Norlin A, Baines DL, Folkesson HG. Role of endogenous cortisol in basal liquid clearance from distal air spaces in adult guinea-pigs. J Physiol (Lond). 1999;519(pt 1):261–272. 31. Sakuma T, Okaniwa G, Nakada T, Nishimura T, Fujimura S, Matthay MA. Alveolar fluid clearance in the resected human lung. Am J Respir Crit Care Med. 1994;150:305–310. 32. Sakuma T, Folkesson HG, Suzuki S, Okaniwa G, Fujimura S, Matthay MA. -Adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am J Respir Cell Mol Biol. 1997;155: 506 –512. 33. Sartori C, Fang X, McGraw DW, Koch P, Snider ME, Folkesson HG, Matthay MA. Selected contribution: long-term effects of 2-adrenergic receptor stimulation on alveolar fluid clearance in mice. J Appl Physiol. 2002;93:1875–1880. 34. Morgan EE, Stader SM, Hodnichak CM, Mavrich KE, Folkesson HG, Maron MB. Postreceptor defects in alveolar epithelial -adrenergic signaling after prolonged isoproterenol infusion. Am J Physiol Lung Cell Mol Physiol. 2003;285:L578 –L583. 35. Morgan EE, Hodnichak CM, Stader SM, Maender KC, Boja JW, Folkesson HG, Maron MB. Prolonged isoproterenol infusion impairs the ability of 2-agonists to increase alveolar liquid clearance. Am J Physiol Lung Cell Mol Physiol. 2002;282:L666 –L674. 36. Liggett SB. Update on current concepts of the molecular basis of 2-adrenergic receptor signaling. J Allergy Clin Immunol. 2002;110: S223–S227. 37. Ricordi C, Shah SD, Lacy PE, Clutter WE, Cryer PE. Delayed extraadrenal epinephrine secretion after bilateral adrenalectomy in rats. Am J Physiol. 1988;254:E52–E3. 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. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/94/8/1091 Data Supplement (unedited) at: http://circres.ahajournals.org/content/suppl/2004/05/12/94.8.1091.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/ 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. Literature Cited 1. 2. 3. 4. 5. 6. Factor P, Senne C, Dumasius V, Ridge K, Jaffe HA, Uhal B, Gao Z, Sznajder JI. Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+ATPase function in A549 cells. Am J Respir Cell Mol Biol. 1998;18:741-9. Factor P, Saldias F, Ridge K, Dumasius V, Zabner J, Jaffe HA, Blanco G, Barnard M, Mercer R, Perrin R, Sznajder JI. Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 subunit gene. J Clin Invest. 1998;102:1421-30. Chruscinski AJ, Rohrer DK, Schauble E, Desai KH, Bernstein D, Kobilka BK. Targeted disruption of the beta2 adrenergic receptor gene. J Biol Chem. 1999;274:16694-700. Rohrer DK, Chruscinski A, Schauble EH, Bernstein D, Kobilka BK. Cardiovascular and metabolic alterations in mice lacking both beta1- and beta2-adrenergic receptors. J Biol Chem. 1999;274:16701-8. Factor P, Saldias F, Ridge K, Dumasius V, Zabner J, Jaffe HA, Blanco G, Barnard M, Mercer R, Perrin R, Sznajder JI. Augmentation of lung liquid clearance via adenoviral-mediated gene transfer of the Na,K-ATPase b1 subunit. J Clin Invest. 1998;102:1142-1150. Hardiman KM, Lindsey JR, Matalon S. Lack of amiloride-sensitive transport across alveolar and respiratory epithelium of iNOS(-/-) mice in vivo. Am J Physiol Lung Cell Mol Physiol. 2001;281:L722-31. 7. 8. 9. 10. 11. 12. 13. 14. Icard P, Saumon G. Alveolar sodium and liquid transport in mice. Am J Physiol. 1999;277:L1232-8. Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, Factor P. b2-Adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats. Circ Res. 2001;89:907-14. Azzam ZS, Dumasius V, Saldias FJ, Adir Y, Sznajder JI, Factor P. Na,K-ATPase overexpression improves alveolar fluid clearance in a rat model of elevated left atrial pressure. Circulation. 2002;105:497-501. Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, Factor P. beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats. Circ Res. 2001;89:907-14. Hollenberg SM, Dumasius A, Easington C, Colilla SA, Neumann A, Parrillo JE. Characterization of a hyperdynamic murine model of resuscitated sepsis using echocardiography. Am J Respir Crit Care Med. 2001;164:891-5. Factor P, Ridge K, Alverdy J, Sznajder J. Continuous enteral nutrition attenuates pulmonary edema in rats exposed to 100% oxygen. J Appl Physiol. 2000;89:1759-65. Kaner RJ, Ladetto JV, Singh R, Fukuda N, Matthay MA, Crystal RG. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema. Am J Respir Cell Mol Biol. 2000;22:657-64. Fang X, Fukuda N, Barbry P, Sartori C, Verkman AS, Matthay MA. Novel role for CFTR in fluid absorption from the distal airspaces of the lung. J Gen Physiol. 2002;119:199-207.