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(Circulation Research. 2001;89:907.)
© 2001 American Heart Association, Inc.

Integrative Physiology

ß₂-Adrenergic Receptor Overexpression Increases Alveolar Fluid Clearance and Responsiveness to Endogenous Catecholamines in Rats

Vidas Dumasius, Jacob I. Sznajder, Zaher S. Azzam, John Boja, Gökhan M. Mutlu, Michael B. Maron, Phillip Factor

From Pulmonary and Critical Care Medicine (V.D., G.M.M., P.F.), Evanston Northwestern Healthcare, Evanston, Ill; Northwestern University Medical School (J.I.S., Z.S.A., G.M.M., P.F.), Chicago, Ill; and Northeastern Ohio Universities College of Medicine (J.B., M.B.M.), Rootstown, Ohio.

Correspondence to Phillip Factor, DO, Pulmonary and Critical Care Medicine, Evanston Northwestern Healthcare, 2650 Ridge Rd, Evanston, IL 60201. E-mail pfactor{at}northwestern.edu

	Abstract

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Abstract—— ß-Adrenergic agonists accelerate the clearance of alveolar fluid by increasing the expression and activity of epithelial solute transport proteins such as amiloride-sensitive epithelial Na⁺ channels (ENaC) and Na,K-ATPases. Here we report that adenoviral-mediated overexpression of a human ß₂-adrenergic receptor (ß₂AR) cDNA increases ß₂AR mRNA, membrane-bound receptor protein expression, and receptor function (procaterol-induced cAMP production) in human lung epithelial cells (A549). Receptor overexpression was associated with increased catecholamine (procaterol)-responsive active Na⁺ transport and increased abundance of Na,K-ATPases in the basolateral cell membrane. ß₂AR gene transfer to the alveolar epithelium of normal rats improved membrane-bound ß₂AR expression and function and increased levels of ENaC ( subunit) abundance and Na,K-ATPases activity in apical and basolateral cell membrane fractions isolated from the peripheral lung, respectively. Alveolar fluid clearance (AFC), an index of active Na⁺ transport, in ß₂AR overexpressing rats was up to 100% greater than sham-infected controls and rats infected with an adenovirus that expresses no cDNA. The addition of the ß₂AR-specific agonist procaterol to ß₂AR overexpressing lungs did not increase AFC further. AFC in ß₂AR overexpressing lungs from adrenalectomized or propranolol-treated rats revealed clearance rates that were the same or less than normal, untreated, sham-infected controls. These experiments indicate that alveolar ß₂AR overexpression improves ß₂AR function and maximally upregulates ß-agonist–responsive active Na⁺ transport by improving responsiveness to endogenous catecholamines. These studies suggest that upregulation of ß₂AR function may someday prove useful for the treatment of pulmonary edema.

Key Words: ß₂-adrenergic receptor • adenovirus • gene transfer • pulmonary edema • alveolar solute transport

	Introduction

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Data from experimental models of lung injury and patients with acute respiratory failure indicate that active alveolar Na⁺ transport is impaired in some forms of pulmonary edema and lung injury.^1–4 Parallel in vitro and in vivo data indicate that this may be due to diminished expression and function of alveolar epithelial transport proteins such as epithelial Na⁺ channels (ENaC) and Na,K-ATPases.^1,2 Other studies indicate that upregulation of these transporters can ameliorate some forms of acute lung injury (ALI) suggesting that there may be a therapeutic role for improving alveolar active Na⁺ transport.^5,6

ß-Adrenergic agonists increase active Na⁺ transport in lung epithelial cells and alveolar fluid clearance (AFC) in animals via stimulation of both ß₁- and ß₂-adrenergic receptors in the alveolar epithelium.^7–10 Stimulation of alveolar epithelial ß₂-adrenergic receptors (ß₂ARs) leads to transcription of solute transport genes, trafficking of assembled solute transporters to the cell membrane, and increased function of membrane-bound epithelial solute transport proteins, all of which can increase AFC.^11–13 The importance of ß-agonists to alveolar solute transport has been shown by Pittet et al,¹⁴ who reported that endogenous catecholamines increase AFC in septic rats. These authors proposed that catecholamine-responsive AFC is an important, protective mechanism that helps keep the lung dry during pathological conditions. Other reports indicate that in some forms of ALI, sepsis, and shock, ß-adrenergic receptor function may be impaired.^15–17

Overexpression of a ß₂AR or a ß-adrenergic receptor kinase (ßARK₁) inhibitor in transgenic mice or via adenoviral-mediated gene transfer in rabbit myocytes and hearts improves ß₂AR function.^18–23 The mechanisms responsible for these findings include attenuation of phosphorylation-induced internalization of membrane bound receptors (downregulation), which allow for maintenance of functional ß₂-receptors in the cell membrane.^18–22 These studies led us to reason that overexpression of a ß₂AR in the alveolar epithelium could improve alveolar ß₂AR function and increase catecholamine-responsive solute transport. To test this hypothesis, we used adenoviral-mediated gene transfer to overexpress a human ß₂AR in human lung epithelial cells and the rat alveolar epithelium. We then tested how overexpression affects receptor function, solute transport protein expression and function, and active Na⁺ transport.

	Materials and Methods

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Adenovirus Propagation and Purification
Replication-incompetent E1a^-/E3^- human type 5 adenoviruses containing either a human ß₂-adrenergic receptor cDNA (adß₂AR, a gift from Drs R. Lefkowitz and W. Koch, Duke University) or no cDNA (adNull) were propagated, purified, and titered as previously described.^24,25 All viruses used in this study were from single preparations and were free of signs of replication-competent adenovirus.

Northern Analysis and rtPCR
Total RNA from A549 cells and lung tissue was isolated using RNA-zol B (Tel-Test Inc, Friendswood, Tex). Northern analysis was performed using a PCR-generated human ß₂AR-specific cDNA probe.²⁴ For rtPCR, RNA was treated with DNAse I prior to first strand cDNA synthesis using oligo-dT. PCR was performed using published primer sequences,²⁰ 30 cycles of amplification, and an annealing temperature of 58°C.

Cell Membrane Isolation
Whole cell membrane proteins were obtained from A549 cells or peripheral lung tissue that was homogenized in (mmol/L): 300 mannitol, 10 Hepes-Tris (pH 7.4) with 3 EGTA/1 EDTA, 0.1 PMSF, and 0.01 mg/mL N-tosyl-L-phenylalanine chlorylmethyl ketone, 0.01 mg/mL leupeptin (homogenization buffer, all from Sigma). Cell debris was removed by centrifugation at 10 000g for 20 minutes at 4°C. The resultant supernatant was twice centrifuged at 100 000g at 4°C, and the resultant pellets resuspended in 100 µL of homogenization buffer. Cell membrane fractions enriched for the apical and basolateral cell membrane domains were produced as described elsewhere.^26,27

cAMP Levels
A549 cells were maintained in serum-free medium for 12 hours prior to study to eliminate the effects of unmeasured catecholamines in fetal bovine serum. Thirty-minutes prior to study, serum-free DMEM containing 1 mmol/L IBMX (10^-3 mol/L, Sigma) was added to limit cAMP degradation. Cellular cAMP levels from cell homogenates were quantified using a radioimmunoassay system (NEN Life Science Products). Mean cAMP values from triplicate experiments are presented as pmol/dish. cAMP production by whole cell membrane fractions (5 to 10 µg of membrane protein) was measured in the presence of Na₂ATP and GTP.²⁸ Production was measured in the presence and absence of procaterol over 30 minutes and is expressed as pmol/mg protein.

Western Blot Analysis
Ten micrograms of membrane protein was separated by 10% SDS-PAGE and transferred to nitrocellulose as previously described.²⁹ A rabbit anti-human ß₂AR antibody (Santa Cruz Scientific, Santa Cruz, Calif), a rabbit anti-human ß₁ Na,K-ATPase antibody (Dr Martin Vasalo, University of Tenerife), a monoclonal anti-human ₁ (Upstate Biotech, Upsala, NY), an immunopurified polyclonal anti-dog ENaC subunit (Dr Douglas Eaton, Emory University), and anti–ß-actin (Santa Cruz Scientific) antibodies were used as primary antibodies. A chemiluminescent system (ECL+, Amersham) was used for immunodetection.

Quantitation of ß₂AR Number
ß-Adrenoreceptor density (Bmax) and affinity for ligand was determined by quantitating binding of ¹²⁵I-cyanopindolol (¹²⁵I-CYP) to cell membrane fractions³⁰ using 5 to 200 pmol/L of ¹²⁵I-CYP. Nonspecific binding of ¹²⁵I-CYP was defined by the addition of 2 µmol/L propranolol for 45 minutes⁵ at 37°C. Bmax and dissociation constants (K_D) were determined using LIGAND. Bmax was normalized to protein used.

Na,K-ATPase Function in A549 Cells
Ouabain-sensitive ⁸⁶Rb⁺ uptake was used to measure Na,K-ATPase function in A549 cells treated with procaterol (10^-6 mol/L) for 10 minutes.²⁴ Specificity of effect was confirmed via addition of the ß₂AR specific antagonist ICI 118,551 (10^-6 mol/L) beginning 10 minutes prior to procaterol treatment. ⁸⁶Rb⁺ data were normalized to untreated, sham-infected controls.

Adenovirus Delivery to Rat Lungs
The use of animals was approved by the Northwestern University and Evanston Northwestern Healthcare Institutional Animal Use and Care Committees. A total of 74 specific pathogen-free adult male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, Ind) were used. Adenovirus was delivered to rats as previously described.²⁴ Briefly, rats were lightly anesthetized and intubated with a 14 g plastic catheter (Angiocath, Becton-Dickenson). While breathing spontaneously, a mixture of adenovirus in a 50% surfactant (Survanta, Abbott Laboratoris)/50% Tris-buffered saline vehicle was administered in 4 aliquots of 0.2 mL via the endotracheal tube. Rats were rotated 90° between instillations given at 5 minute intervals. To facilitate distal dispersion of adenovirus, forced exhalation was achieved by compression of the thorax, vehicle was then instilled (followed by 0.5 mL of air), and compression was relinquished. Infected animals were studied 7 days after vector delivery.

Na,K-ATPase Function (P_i Liberation From ATP) in BLMs
Twenty micrograms of BLM protein was resuspended in 100 µL of a high [Na⁺]/low [K⁺] reaction buffer (in mmol/L: 50 Tris-HCl pH 7.4, 50 NaCl, 5 KCl, 10 Mg₂Cl, 1 mmol/L EGTA, 10 mmol/L Na₂ATP, with [-³²P]-ATP [3.3 nCi/µL]). Triplicate samples were placed at -20°C for 15 minutes prior to incubation for 15 minutes at 37°C. The reaction was terminated by addition of 5% TCA/10% charcoal and cooling to 4°C. The charcoal phase containing unhydrolyzed nucleotide was separated by centrifugation (12 000g for 5 minutes) and the liberated ³²P quantified. Na,K-ATPase activity was calculated as the difference between the test samples (total ATPase activity) and samples assayed in reaction buffer with 2.5 mmol/L ouabain but devoid of Na⁺ and K⁺ (nonspecific ATPase activity). Results are expressed as nmol of P_i/mg of protein/hour.

Isolated Lung Experiments
Briefly, known concentrations of ²²Na⁺, ³H-manitol, and Evans Blue–tagged albumin are instilled into the airspace of isolated lungs as previously described.⁶ Fluid samples from the airspace were obtained at the start of each isolated lung study and after 60 minutes. Changes in alveolar Evan’s blue concentration were used to calculate changes in volume of alveolar fluid. Movement of tracers out of the airspace and into the vasculature was used to measure alveolar permeability.

Treatment Protocols
Alveolar permeability and clearance were measured in sham-, adNull-, and adß₂AR-infected rats (untreated=5 animals/group, amiloride treated=4 animals/group). The effects of procaterol on AFC were determined in uninfected (n=3) and adß₂AR-infected (n=3) rat lungs. To block the effects of endogenous catecholamines, sham- or adß₂AR-infected rats (6 animals/group) were treated with propranolol (1 mg/kg every 8 hours by gavage) for 5 days beginning 2 days after infection. Adrenalectomized rats (n=4 animals/group) were provided with 0.9% NaCl for drinking, per supplier’s recommendations. Procaterol (10^-6 mol/L, administered via the instillate and perfusate) and amiloride (10^-6 mol/L, administered via the alveolar instillate only) were added beginning 10 minutes prior to the isolated lung studies.

Immunohistochemistry
Longitudinal lung sections were treated with 3% H₂O₂ to reduce endogenous peroxidase activity prior to blocking of nonspecific immunoreactivity with nonimmune goat serum.^6,24 Rabbit anti-human ß₂AR antibody was added for 1 hour at 25°C prior to washing and immunodetection using a fluorescence-linked secondary antibody.

Data Analysis
All values are reported as means±standard deviation. Statistical comparison among groups was performed using one-way ANOVA (DataDesk, Data Description). Statistical significance was defined as P<0.05.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

	Results

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Adenoviral-Mediated ß₂AR Gene Transfer Increases Receptor Number and Function in Human Lung Epithelial Cells Transduced With adß₂AR
Human ß₂AR mRNA was detected using rtPCR (data not shown) but not northern analysis (Figure 1A) in sham- and adNull-infected controls, which is consistent with the low-level expression of this gene in these cells,³¹ whereas ß₂AR protein was noted only in cells infected with adß₂AR (Figure 1B). Membrane-bound receptor number (¹²⁵I-CYP binding) in adß₂AR-infected cells was nearly 500% greater than adNull-infected cells (7.7 fmol/mg protein and 1.6 fmol/mg protein, respectively). Scatchard analysis of ¹²⁵I-CYP binding revealed no difference in receptor affinity between adß₂AR- and adNull-infected cells (23.3 pmol and 27.5 pmol, respectively).

View larger version (54K): [in this window] [in a new window]   Figure 1. Transgene expression and function in A549 cells. A, Northern and (B) Western analyses of sham-, adNull-, and adß2AR-infected A549 cells. C, cAMP levels in cell lysates from cells treated with procaterol. Data (mean±standard deviation) is pmol/dish. *P<0.002 vs untreated adß2AR, untreated adNull, and procaterol-treated adNull; **P<0.01 vs untreated adNull and untreated adß2AR.

Baseline receptor function, assayed by measuring cAMP levels in cell lysates after 12 hours of serum deprivation, was not different in adß₂AR- (29±6.4 pmol/dish) and adNull (22.1±5.3 pmol/dish) infected cells (Figure 1C). Addition of the ß₂-specific agonist, procaterol (10^-6 mol/Lx1 minute) increased cAMP levels in adß₂AR cells by 11-fold to 328.9±36.4 pmol/dish (Figure 1C) to nearly the same level as adß₂AR-infected cells treated with the adenylate cyclase activator, forskolin (348±24.7 pmol/dish). Procaterol increased cAMP levels in adNull controls to a lesser degree (5-fold to 118.3±29.7 pmol/dish).

ß₂AR Overexpression Increases Na,K-ATPase Activity and Membrane-Bound Abundance in A549 Cells
To determine if ß₂AR overexpression affects catecholamine-responsive active Na⁺ transport, we measured ouabain-sensitive ⁸⁶Rb⁺ uptake. Uptake in serum-deprived, untreated cells was not different between sham-, adNull-, or adß₂AR-infected cells (Figure 2A). The addition of procaterol (10^-6 mol/Lx10 minutes) increased uptake by up to 250% in cells infected with adß₂AR and to a lesser in sham- and adNull-infected cells. The effects of procaterol were blocked by pretreatment with the ß₂AR specific antagonist ICI 118,551 (10^-6 mol/Lx10 minutes, Figure 2A) in all groups confirming that these observations are due to ß₂-receptor function and not nonspecific effects of adenoviral-mediated gene transfer.

View larger version (44K): [in this window] [in a new window]   Figure 2. Effect of ß2AR overexpression on active transport and solute transport proteins in A549 cells. A, Procaterol responsive ouabain-sensitive 86Rb+ uptake (Na,K-ATPase activity). Data (mean±standard deviation) is normalized to untreated, sham-infected control. *P<0.05 vs untreated and ICI 118,551 treated controls; **P<0.02 vs all other groups. B, Western analysis of Na,K-ATPase 1 and ß1 subunit abundance in BLMs isolated from A549 cells infected with adNull or adß2AR and treated with procaterol for 10 minutes. Densitometric quantitation of Na,K-ATPase subunit protein levels from 3 separate experiments/condition (graph) and representative Western blots for ß1 (upper blot) and 1 (lower blot) are shown. Data are normalized to untreated, sham-infected controls and are presented as mean±standard deviation. *P<0.05 adß2AR-infected vs all other groups.

To determine if overexpression affects levels of membrane-bound Na,K-ATPase subunit proteins, we isolated basolateral membrane (BLM) fractions from adß₂AR- and adNull-infected A549 cells that were treated with procaterol (10^-6 mol/Lx10 minutes). Western analysis (Figure 2B) of these membranes shows significantly greater Na,K-ATPase ₁ and ß₁ subunit abundance in BLMs from adß₂AR cells than similarly treated adNull controls.

Gene Transfer Increases ß₂AR Abundance and Function in the Peripheral Lung of Rats
To test if gene transfer could affect ß₂AR overexpression in the alveolar epithelium, we infected rats with 4x10⁹ pfu (4.7x10¹¹ viral particles) of adenovirus using an established surfactant-based delivery system that is capable of widespread gene transfer in rat lungs.²⁴ All animals were studied 7 days after infection to allow vector-induced host responses to subside. In preliminary dose response studies it was noted that doses greater than this were associated with lung injury and occasional death. Human ß₂AR was detected via rtPCR, Western analysis, and immunohistochemistry only in lungs infected with adß₂AR (n=3 rats/group, Figure 3). The high-power photomicrograph included in the inset in Figure 3C shows a linear pattern of immunofluorescence that parallels the airspace (Figure 3C), indicating that our delivery system transduces the alveolar epithelium.

View larger version (31K): [in this window] [in a new window]   Figure 3. Transgene expression in rat lungs. A, Reverse-transcriptase PCR and (B) Western analyses of whole lung homogenates for human ß2AR signal 7 days after. C, Representative photomicrographs of rat lungs immunostained with an anti-human ß2AR antibody. The inset in the right photomicrograph is a high-power (1000x) view of adß2AR-infected lungs; arrows identify areas of linear immunofluorescence that parallel the alveolar airspace.

To determine if our transgene was functional in vivo, we harvested cell membranes from peripheral lung tissue (to diminish the contribution of airway ß₂ARs) and measured cAMP production and expression. Human ß₂AR was noted only in membranes isolated from lungs infected with adß₂AR (n=3/group) (Figure 4A). Rat ß₂AR abundance in adß₂AR-infected lungs was not different from sham and adNull controls. Procaterol-responsive cAMP production (ß₂AR function) by membrane fractions from adß₂AR-infected lungs was 450% greater than similarly treated membranes from sham-infected lungs (Figure 4B). Catecholamine-responsive cAMP production by membranes from adNull-infected lungs was minimally greater than sham-infected controls (P=0.05 versus procaterol treated sham-infected membranes) and likely represents vector/infection related effects.

View larger version (58K): [in this window] [in a new window]   Figure 4. Transgene expression and function in peripheral lung cell membranes. A, Representative lanes from a single Western blot of whole-cell membrane fractions isolated from peripheral lung tissue for human and rat ß2AR. ß-actin is included to demonstrate uniformity of lane loading. B, Baseline (untreated) and procaterol-responsive cAMP production by whole-cell membrane fractions isolated from peripheral lung tissue. *P<0.02 procaterol treated vs untreated membranes from sham-infected, untreated membranes; **P<0.0015 vs all other groups.

Receptor Overexpression Improves Alveolar Fluid Clearance in Rat Lungs and Increases the Expression and Function of Solute Transport Proteins on Both Sides of the Cell
To determine whether ß₂AR overexpression affects vectorial Na⁺ movement in the alveolus, we used an isolated, perfused rat lung preparation that allows measurement of alveolar fluid clearance (AFC) and permeability.³² Clearance in unstimulated adß₂AR-infected rats was increased (1.37±0.09 mL/hour, n=5) as compared with uninfected controls treated with procaterol (1.00±0.04 mL/hour, n=3), adNull-infected (0.68±0.18 mL/hour, n=5) and sham-infected controls (0.73±0.02 mL/hour, n=5) (Figure 5A). The addition of procaterol (10^-6 mol/L, n=3) to adß₂AR lungs did not increase AFC (1.24±0.27 mL/hour), further suggesting that receptor overexpression maximally increases ß₂AR-responsive AFC (Figure 5B). Alveolar permeability for Na⁺ and mannitol was minimally increased in adß₂AR rats (Figures 5C), permeability for albumin was not different among the groups. Permeability in procaterol-treated lungs was not different from untreated lungs.

View larger version (17K): [in this window] [in a new window]   Figure 5. Alveolar fluid clearance and permeability. A, Alveolar fluid clearance in uninfected rat lungs treated with procaterol (10-6M, n=3) during isolated lung studies (n=5 rats/group). B, Effect of the addition of procaterol to the alveolar instillate and vascular perfusate of adß2AR-infected lungs (n=3 rats/group). C, Movement of Na+ and mannitol between airspace, vascular, and pleural compartments (an index of alveolar permeability). Data are mean±standard deviation. *P<0.01 uninfected controls treated with procaterol vs sham-, adNull-, and adß2AR-infected lungs; **P<0.01 vs all other groups; ***P=0.05 adß2AR vs sham- and adNull-infected lungs.

To determine if ß₂AR overexpression affects epithelial Na⁺ channel (ENaC) function, amiloride (10^-6 mol/L) was instilled into the airspace beginning 10 minutes prior to measurement of AFC. In preliminary experiments, it was noted that higher doses of amiloride (10^-4 mol/L, n=4) completely stopped AFC, probably by inhibiting both ENaC and non-ENaC solute transporters. Amiloride (10^-6 mol/L, Figure 6A) reduced clearance by 80% in adß₂AR lungs (0.26±0.06 mL/hour, n=4), 40% in sham (0.28±0.07 mL/hour, n=4), and 42% in adNull (0.26±0.03 mL/hour, n=4) lungs suggesting that amiloride-sensitive Na⁺ uptake is upregulated in adß₂AR-infected lungs. Permeability for Na⁺, mannitol, and albumin in lungs treated with amiloride was not different from untreated lungs.

View larger version (18K): [in this window] [in a new window]   Figure 6. Membrane bound solute transport protein abundance. A, Changes in amiloride-responsive AFC (n=4/group). *P<0.02 adß2AR-infected vs sham and adNull. B, Na,K-ATPase activity (ouabain-sensitive Pi hydrolysis measured under conditions of Vmax) in BLMs from the peripheral lung of rats treated with and without propranolol (n=3 rats/group). *P<0.002 adß2AR vs sham and adNull. C, Western analysis for epithelial Na+ channel subunit (ENaC) abundance in APM fractions (20 µg protein/lane) isolated from peripheral lung tissue of rats treated with and without propranolol (n=3 rats/group). Graphical data represent mean density of an 85 kDa band normalized to sham-infected control in the western blot shown. *P<0.02 adß2AR vs sham and adNull. Peripheral lung tissue from the same 3 rats was used for both Na,K-ATPase activity and ENaC quantitation. All data are mean±standard deviation.

We next assessed how ß₂AR overexpression affects solute transport proteins by measuring Na,K-ATPase activity (ouabain-sensitive liberation of P_i from ATP) in BLMs isolated from the peripheral lung. The conditions used in these experiments eliminate cation concentration-dependent effects on enzyme function and produce an index of functional, membrane-bound Na,K-ATPase number. As compared with sham- and adNull-infected controls, Na,K-ATPase activity in BLMs from adß₂AR-infected lungs was increased by >300% (n=3 lungs/group, Figure 6B). Similarly, Western analysis of membrane protein fractions enriched for the apical membrane (APM) domain revealed increased levels of ENaC ( subunit) in rats infected with adß₂AR (n=3 lungs/group, Figure 6C). No Na,K-ATPase ₁ or ß₁ subunit protein was noted in APM preparations from any group. Na,K-ATPase number (Figure 6B) and ENaC (Figure 6C) expression in membranes from rats treated with the nonspecific ß-blocker propranolol for 5 days were not different from controls.

Receptor Overexpression Improves Responsiveness to Endogenous Catecholamines
To control for the effects of endogenous catecholamines in our model, we treated rats with the nonspecific ß-blocker propranolol for five days prior to measurement of AFC. Clearance in propranolol-treated/adß₂AR-infected rats (0.41±0.24 mL/hour, n=3) was not different from propranolol-treated, sham-infected controls (0.42±0.09 mL/hour, n=3) (Figure 7A). However, AFC in these rats was less than untreated, sham-infected controls (P<0.02, propranolol treated adß₂AR and sham versus untreated sham-infected controls). To control for possible negative inotropic effects of propranolol, we also studied AFC in rats with reduced levels of serum catecholamines due to adrenalectomy (Figure 7B). Like propranolol-treated rats, clearance in sham-infected adrenalectomy lungs was reduced by 40% (0.45±0.04 mL/hour, n=4) as compared with normal, sham-infected rats (Figure 5A). Fluid clearance in adrenalectomized, adß₂AR-infected rats (0.71±0.09 mL/hour, n=4) was greater than in sham-infected adrenalectomized rats (0.45±0.05 mL/hour) but was not different from untreated/normal, sham-infected controls (0.73±0.02 mL/hour). Clearance in adrenalectomized adß₂AR rats was significantly less than normal adß₂AR-infected rats (0.71±0.09 mL/hour versus 1.37±0.09 mL/hour). Treatment of these lungs with procaterol (10^-6 mol/L) for 60 minutes following completion of the initial clearance measurements increased AFC to 1.13±0.27 mL/hour and 1.07±0.13 mL/hour in adß₂AR- and sham-infected controls, respectively, confirming that these lungs possess functional ß-receptors. Permeability for large and small solutes in sham- and adß₂AR-infected adrenalectomized or propranolol-treated rat lungs was not different from normal and untreated sham- and adß₂AR-infected lungs.

View larger version (23K): [in this window] [in a new window]   Figure 7. Effect of endogenous catecholamines. AFC was measured in sham and adß2AR-infected lungs from (A) rats treated with propranolol for 5 days (n=3 rats/group) and (B) adrenalectomized rats (n=4 rats/group). Procaterol was added to lungs from adrenalectomized rats following initial clearance measurements to confirm if these rats have functional ß2ARs. *P<0.02 vs sham-infected, untreated adrenalectomy group.

To test how attenuation of receptor signaling affects Na⁺ transport protein expression apical and basolateral cell membranes were isolated from the peripheral lung of propranolol-treated, ß₂AR overexpressing lungs and used to quantitate levels of ENaC (Western analysis) and Na,K-ATPase (P_i hydrolysis). As can be seen in Figure 6, membrane-bound levels of these proteins were not different from sham-infected controls indicating that the upregulation of these proteins noted in normal adß₂AR-infected lungs is due to receptor signaling and not nonspecific effects of gene transfer.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A broad base of data indicates that impairment of AF contributes to pulmonary edema and that methods that improve Na⁺ transport may be useful in attenuating lung injury.⁶ ß-Adrenergic agonists have been shown to increase active Na⁺ transport in alveolar epithelial cells, in normal and injured animal lung, and human lung explants (reviewed in Berthiaume³³). These reports suggest a role for these sympathomimetics in the treatment of pulmonary edema. Studies from cardiac myocytes and rabbit hearts indicate that overexpression of a ß₂AR can improve ß₂AR function and prevent receptor desensitization.^20,21 These reports caused us to hypothesize that adenoviral-mediated overexpression might similarly improve alveolar ß₂-receptor function and increase catecholamine-responsive active Na⁺ transport.

The in vitro experiments in this study confirm that our vector is functional in lung cells and highlight the potential of adenoviral-mediated gene transfer for increasing receptor expression and cAMP production (Figures 1 and 2). The findings of increased active Na⁺ transport and increased levels of membrane bound Na,K-ATPase (Figure 2) provide mechanistic information that suggests that augmentation of ß₂-receptor function increases solute transporter abundance in the appropriate domain of the cell membrane. The duration of procaterol stimulation used in our study (10 minutes) makes new mRNA or protein synthesis an unlikely explanation for this data. A previous study has shown that catecholamines (isoproterenol) increase Na,K-ATPase activity by recruiting assembled Na,K-ATPases from early endosomes to the plasma membrane.³⁴ Our data suggest that this may also occur in the current model and contribute to the observed increases in Na,K-ATPase activity.

Our in vitro experiments are paralleled by data from rat lungs showing that overexpression increases ß₂AR expression and function (Figure 4) in cell membranes harvested from the peripheral lung. We used peripheral lung tissue to limit the contributions of bronchial epithelial and smooth muscle cells to our measurements of ß₂AR function. Thus, we believe that our complementary data from peripheral (Figure 4) and whole lung (Figure 3) provide support for the hypothesis that receptor overexpression increases the expression and function of ß₂ARs in the cell membrane of alveolar cells.

To pursue the mechanisms responsible for increased AFC in adß₂AR-infected animals, we conducted isolated lung studies using the Na⁺ channel blocker amiloride. When used in adß₂AR rats, amiloride (10^-6 mol/L) reduced AFC to a greater degree than in sham and adNull rats (80% versus 40%) suggesting that amiloride-sensitive Na⁺ transport is increased in our model (Figure 5A). This finding is corroborated by data showing increased ENaC expression in APMs collected from adß₂AR-infected lungs (Figure 6B). These data, combined with increased functional Na,K-ATPases in BLMs (Figure 6A), suggest that increased ß₂AR function upregulates both apical (ENaC) and basolateral (Na,K-ATPase) transport pathways. We believe it reasonable to speculate that this upregulation contributes to the increased AFC noted in the current study (Figure 5A). Data from propranolol-treated rats (Figures 6B and 6C), showing that receptor blockade prevents changes in membrane bound ENaC and Na,K-ATPase activity, indicate that our findings are due to increased receptor signaling and not nonspecific effects of adenoviral-mediated gene transfer.

The finding of increased AFC without exogenous catecholamine supplementation (Figure 5A) and the failure of procaterol to increase AFC further in adß₂AR lungs (Figure 5B) suggests that the increased AFC in adß₂AR animals may be due to improved responsiveness to endogenous (serum) catecholamines. Thus, we conducted additional experiments to control for endogenous catecholamines by using adrenalectomized animals and the ß-blocker propranolol. These experiments revealed AFC rates that were less than normal/untreated adß₂AR-infected lungs (Figures 7A and 7B). Similarly, membrane bound ENaC levels and Na,K-ATPase activity from these animals was not different between sham- and adß₂AR-infected lungs (Figure 6). Both of these approaches have experimental limitations including negative inotropic effects due to ß-receptor blockade or diminished expression of solute transport proteins due to reduced serum catecholamine and steroidal hormone levels following adrenalectomy. However, we believe that data from these complementary approaches allows us to conclude that receptor overexpression augments responsiveness to endogenous catecholamines and increases the abundance of membrane bound Na⁺ transport proteins, which contribute to the increases in AFC noted in ß₂AR overexpressing lungs (Figure 5A).

Our findings of increased receptor function (Figure 4) and AFC (Figure 5) in ß₂AR overexpressing lungs in the absence of catecholamine supplementation raises the possibility that our transgene may be constitutively active in rats. Our finding that ICI 118,551 blocks procaterol-mediated increases in active Na⁺ transport in serum-starved A549 cells (Figure 2) and our results from propranolol-treated and adrenalectomized rats (Figures 7) suggest otherwise. Similarly, mice that overexpress a ß₂AR in alveolar type 2 epithelial cells have increased AFC and Na,K-ATPase (₂ subunit) expression that can be attenuated by adrenalectomy.²³ Another hypothesis for the observed increases in receptor function due to overexpression can be found from prior studies of receptor overexpression in cardiac myocytes from failing rabbit hearts.^19,20,35 These studies show that receptor overexpression attenuates agonist-induced receptor desensitization by maintaining functional receptors in the cell membrane. Thus, the increased basal (ie, unstimulated) ß₂AR function noted in the current study and that of McGraw et al²³ may represent inability of alveolar cells to upregulate endogenous phosphorylation mechanisms that would be expected to curtail cAMP production in the setting of receptor overexpression. Although, investigations into how overexpression affects alveolar ß₂AR desensitization represent an important area for future investigation, it is important to emphasize that in the current study overexpression positively affected AFC regardless of whether agonist-induced loss of receptor function occurs in the alveolus.

This study demonstrates that ß₂AR overexpression can positively affect ß₂-adrenergic receptor function in lung cells and the alveolar epithelium. The findings of increased membrane-bound Na⁺ transport protein expression and function and improved responsiveness to endogenous catecholamines indicate that ß₂AR overexpression enhances alveolar active Na⁺ transport by upregulating solute transport pathways on both sides of the cell. We believe that our data, which has been generated using complementary in vitro and in vivo experiments, support the concept that ß₂ARs are important modulators of alveolar active Na⁺ transport. Early generation adenovectors, such as those used in this study, cause innate and acquired host-responses which limit their use in humans. However, newer adenovectors, which are devoid of adenovirus genes,³⁶ combined with masking of capsid epitopes³⁷ offers the possibility that adenoviral-mediated gene transfer could eventually prove safe for treatment of human lung diseases. The data presented herein suggest that upregulation of ß₂AR function, via gene transfer, may someday be an option for the treatment of pulmonary edema.

	Acknowledgments

 
This work was supported by the American Heart Association, the Evanston Northwestern Healthcare Research Institute, HL-48129, and HL-66211.

Received May 8, 2001; revision received October 2, 2001; accepted October 2, 2001.

	References

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