Targeted transgenic expression of beta 2-adrenergic receptors to type II cells increases alveolar fluid clearance

Dennis W. McGraw1,*, Norimasa Fukuda2,*, Paul F. James3,*, Susan L. Forbes1, Alison L. Woo4, Jerry B. Lingrel4, David P. Witte5, Michael A. Matthay2, and Stephen B. Liggett1,3

Departments of 1 Medicine and 4 Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati 45267, 3 Department of Zoology, Miami University, Oxford 45056; 5 Department of Pathology, Children's Hospital Medical Center, Cincinnati, Ohio 45229; and 2 Cardiovascular Research Institute, University of California, San Francisco, California 94143


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta -agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta 2-adrenergic receptors (beta 2-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta 2-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta 2-ARs than nontransgenic (NTG) mice (939 ± 113 vs. 194 ± 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was ~40% greater than that in untreated NTG mice (15 ± 1.4 vs. 10.9 ± 0.6%; P < 0.005) and approached that of NTG mice treated with the beta -agonist formoterol (19.8 ± 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 ± 0.5% but had no effect on NTG mice (11.5 ± 1.0%). Na+-K+-ATPase alpha 1-isoform expression was unchanged, whereas alpha 2-isoform expression was ~80% greater in the TG mice. These findings show that beta 2-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta 2-ARs specifically expressed on ATII cells.

G protein; adenylyl cyclase; beta -agonist; mouse; pulmonary edema


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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THE ALVEOLAR EPITHELIUM FORMS a critical barrier against the movement of water into the alveolar space and plays an important role in the active removal of excess alveolar fluid and protein. This reabsorption of excess fluid is driven by the vectorial transport of sodium ions across the alveolar epithelium (reviewed in Ref. 21). Sodium ion uptake probably occurs primarily across alveolar type II (ATII) cells where it enters the cell through amiloride-sensitive and amiloride-insensitive channels on the apical surface (20). Sodium ions are then actively pumped from the basolateral surface of the ATII cell by Na+-K+-ATPase (28). Clearance of alveolar fluid (AFC) results from the passive movement of water that subsequently follows the active transport of sodium ions and perhaps chloride (17).

Endogenous catecholamines and exogenously administered beta -agonists can accelerate the removal of excess fluid from the alveolar space. These effects have been observed in vivo for several different mammalian species (2, 3, 12) and ex vivo in human resected lung (34, 35). It has also been shown in animal models that the enhanced clearance of pulmonary edema fluid after neurological insult (16), hemorrhage (27, 30), and sepsis (31) is dependent on the release of endogenous catecholamines. Activation of the beta 2-adrenergic receptor (beta 2-AR) on alveolar epithelial cells may therefore serve a protective function that limits alveolar flooding and enhances its resolution. This has led to the proposal that beta -agonist therapy could be a possible treatment for patients with acute pulmonary edema (21, 40).

The effects of beta -agonists on lung function in intact animals are potentially confounded by the multiple cell types that express beta 2-ARs. The receptor is expressed on airway epithelium and smooth muscle and vascular endothelium and smooth muscle as well as on one or more cell types that line the alveolus (6, 10). The cell-specific effects of beta 2-AR activation on AFC, particularly those of the vasculature and alveolar epithelium, may therefore be difficult to distinguish when functional studies are being done. Recently, McGraw et al. (24) and others (25, 39) have shown that beta 2-AR overexpression can stimulate the signaling cascade in vivo by increasing the pool of spontaneously activated receptor and/or enhancing the sensitivity to endogenous agonists. Receptor activation can be limited to a specific cell type via transgenesis by directing expression with a cell-specific promoter, thereby permitting the in vivo effects of beta 2-AR signaling in a given cell to be differentiated from those of other cell types within the same environment (24). In the present study, we generated transgenic mice that selectively overexpressed the beta 2-AR in ATII cells. Our goal was to distinguish the effects of beta 2-AR activation in ATII cells on AFC in intact mice from those of other cells in the alveolus and to delineate a mechanism by which this effect might occur. In addition, potential untoward effects of such overexpression on lung development and function were assessed.


    METHODS
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INTRODUCTION
METHODS
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Transgenic mice. Expression of the human beta 2-AR was directed to ATII cells with the promoter from the rat surfactant protein (SP) C gene (14). To construct the SP-C-beta 2-AR transgene, a 3.7-kb HindIII-HindIII fragment from the rat SP-C promoter (a gift from Dr. J. Whitsett, Children's Hospital Medical Center, Cincinnati, OH), a 1.5-kb HindIII-PshAI fragment encoding the human beta 2-AR open reading frame (ORF), and a 0.85-kb XhoI-BamHI fragment encoding the SV40 small t intron and polyadenylation signal were ligated together in the vector pUC18. The orientation of each fragment was confirmed by sequence analysis and restriction enzyme digestion. The 6.05-kb transgene was released from the vector by NotI digestion, gel purified, and dialyzed against 5 mM Tris · HCl (pH 7.4) and 0.1 mM EDTA. The purified DNA was then injected into fertilized eggs of FVB/N mice, and the eggs were implanted into pseudopregnant females with methods previously described (39). Founder mice were identified by Southern blot analysis of genomic DNA prepared from tail clips. Independent lines of heterozygous SP-C-beta 2-AR mice were maintained by mating the transgenic mice with nontransgenic FVB/N mice. Subsequent screening for the heterozygous progeny was by PCR analysis of the genomic DNA with a forward primer from the beta 2-AR ORF (5'-GGAGCAGAGTGGATATCACG-3') and a reverse primer from the SV40 polyadenylation region (5'-GTCACACCACAGAAGTAAGG-3'). Heterozygous mice from generations 2 to 5 between the ages of 8 and 16 wk were used for all studies.

Transgene expression and localization. To assess transgene expression among the independent transgenic lines, RNase protection assays (RPAs) were performed with a 32P-labeled antisense riboprobe corresponding to the distal 500 bp of the human beta 2-AR ORF as previously reported (23). McGraw et al. (24) have previously shown that this probe does not recognize the endogenous mouse beta 2-AR transcript. Lung RNA was prepared from lungs with TRI Reagent (Molecular Research Center, Cincinnati, OH). For the RPA, RNA (20 µg) and the beta 2-AR riboprobe were hybridized overnight, digested with RNase, and subjected to denatured PAGE analysis. A radiolabeled antisense riboprobe for mouse beta -actin was included in each reaction to serve as an internal positive control and to account for differences in gel loading. The gels were visualized with a phosphorimager (Molecular Dynamics) and analyzed with the ImageQuant software package (Molecular Dynamics).

The distribution of transgene expression within the lung was assessed with in situ hybridization as previously reported (41). Briefly, the lungs were rapidly dissected, fixed in 4% paraformaldehyde, cryoprotected with 30% sucrose in PBS, and frozen in optimum cutting temperature compound. Cryostat sections (7 µm) were then mounted on silane-coated slides. An antisense cRNA probe for the human beta 2-AR was prepared as described above for the RPA studies except that the probe was labeled with 35S-UTP. A sense cRNA probe was prepared with SP6 polymerase for use as a negative control. Hybridization was performed with 0.5-1.0 × 106 counts/min of labeled probe in a final volume of 30 µl/slide. After overnight incubation at 42°C, the sections were treated with 50 µg/ml of RNase A and 100 U/ml of RNase T1 for 30 min at 37°C and washed to a final stringency in 0.1× saline-sodium citrate at 50°C. The slides were dipped in NTB2 emulsion (Eastman Kodak) diluted 1:1 with 0.6 M ammonium acetate and exposed for 2 wk, after which they were developed with D19 developer (Eastman Kodak) and counterstained with hematoxylin and eosin.

Receptor density and adenylyl cyclase. Lung membranes were prepared from individual mice by homogenizing the entire lung or trachea in 10 ml of hypotonic lysis buffer (5 mM Tris, pH 7.4, and 2 mM EDTA) containing the protease inhibitors leupeptin, aprotinin, benzamidine, and soybean trypsin inhibitor (10 µg/ml each). The homogenates were centrifuged at 40,000 g for 10 min at 4°C. The supernatant was removed, and the pellets containing the crude membrane particulates were suspended in assay buffer (75 mM Tris, pH 7.4, 12.5 mM MgCl2, and 2 mM EDTA). Receptor expression was determined by radioligand binding with [125I]iodocyanopindolol as previously described (23).

Adenylyl cyclase activity in these membrane preparations was assessed with a column chromatography method as previously reported (22).

AFC studies. The mice were killed by an overdose of pentobarbital sodium (200 mg/kg ip), and a 20-gauge trimmed angiocath plastic needle (Becton Dickinson) was inserted into the trachea. The lungs were then inflated with 7 cmH2O continuous airway pressure with 100% oxygen throughout the experiment. An infrared lamp placed 30 cm above the body was cycled on and off to maintain body temperature at 37°C. Body temperature was monitored by placing a temperature probe (Yellow Springs Instruments) into the abdominal cavity via a 0.5-cm incision. In some experiments, bilateral adrenalectomies were performed 8-12 h before the AFC studies.

AFC was measured as previously reported (2, 9, 12, 19). The lungs were instilled with Ringer lactate containing 5% bovine serum albumin and 0.1 µCi of 131I-albumin (Merck-Frosst, Montreal, PQ) as an alveolar protein tracer. Osmolarity of the instillate was adjusted to 340 mosM, which we have shown to be isosmolar with mouse plasma (9). In some experiments, different concentrations of the beta 2-selective agonist formoterol were added to the instillate. AFC was measured over 15 min. As in previous studies by our laboratory (2, 9, 19), AFC was determined by measuring the increase in the final concentration of the alveolar protein tracer compared with the initial instilled tracer protein concentration. AFC was calculated as AFC = [(Vi - Vf)/Vi] × 100, where Vi is the volume of the initial alveolar fluid and Vf is the volume of the final alveolar fluid. Vf was calculated as Vf = (Vi × Tpi × Fr)/Tpf, where Tpi is the initial total tracer protein concentration in the alveolar fluid, Tpf is the final total tracer protein concentration in the alveolar fluid, and Fr is the fraction of alveolar tracer protein that remains in the lung at the end of the experiment.

Western blot analysis. The expression of Na+-K+-ATPase alpha 1- and alpha 2-isoforms in microsomal membrane preparations was assessed by Western blot analysis as previously reported (11). For each sample, whole lungs from four mice were homogenized in 7 ml of buffer (250 mM sucrose, 30 mM histidine, pH 7.2, and 2 mM EDTA) with two 30-s bursts of a Polytron homogenizer. Microsomes were prepared from these homogenates as previously reported except that NaI treatment was omitted (13). For Western blotting, the samples were incubated for 30 min at 37°C in 50 mM Tris (pH 6.9), 5% SDS, 1% beta -mercaptoethanol, and 10% glycerol. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After electrophoresis, the gels were transferred overnight to polyvinylidene difluoride membranes. The blots were blocked for 1 h at room temperature in 5 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 that contained 1% blocking reagent (Boehringer Mannheim). The blots were then incubated for 1 h at room temperature in 5 mM Tris, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 containing either the Na+-K+-ATPase alpha 1-isoform-specific monoclonal antibody alpha 6f (University of Iowa Developmental Hybridoma Bank, Iowa City) or the alpha 2-isoform-specific monoclonal antibody McB2 (provided by Dr. Kathleen Sweadner, Massachusetts General Hospital, Charlestown, MA). Afterward, the blots were washed and then incubated with a peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch). Immunoreactivity was visualized with an enhanced chemiluminescence system (Amersham Life Sciences) and Kodak BioMax MR X-ray film. Exposure times were individually optimized to provide the maximal signal intensity for each isoform. The resulting signals were quantified by densitometry with ImageQuant software (Molecular Dynamics). Two microsomal preparations, each composed of four pooled lungs, were analyzed for each group. Each blot contained multiple protein concentrations (30, 60, and 120 µg) to ensure linearity of the signal.

Statistical analysis. Data are reported as means ± SE. Statistical comparisons between the nontransgenic and transgenic groups were performed with a two-tailed Student's t-test. Differences were considered significant at P < 0.05.


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ABSTRACT
INTRODUCTION
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Transgenic mice. Southern blot analysis was used to screen for SP-C-beta 2-AR founder mice. Figure 1A shows that the transgene was detected in 5 of 20 mice screened. To generate transgenic lines, founder mice (designated 2.2, 3.1, 3.2, 4.3, and 5.2) were mated with nontransgenic FVB/N mice. Transgenic progeny were detected in all lines except those from founder 3.2. For each transgenic line established, the transgene was inherited in ~50% of the progeny with an equal distribution between males and females. Growth, development, and survival of mice from each transgenic line were not different from those of nontransgenic littermates. Histological examination of the lungs as well as of other organs from SP-C-beta 2-AR mice was unremarkable.


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Fig. 1.   Transgene detection and expression in surfactant protein (SP) C-beta 2-adrenergic receptor (beta 2-AR) mice. A: genomic DNAs from 5 founder mice (nos. on top; +) were detected by Southern blot analysis. -, Nontransgenic (NTG) mice. B: RNase protection assay with a human-specific beta 2-AR antisense riboprobe showed that significant transgene expression was present in lines generated from only 2 of the founders (2.2 and 4.3). Nos. at left, bp.

Transgene expression and localization. To confirm that the SP-C-beta 2-AR transgene was being expressed in each transgenic line, total cellular RNA was subjected to RPAs. RNA was hybridized with an antisense riboprobe corresponding to the distal 500 bp of the human beta 2-AR. McGraw et al. (23) have shown that this probe is specific for the human receptor and does not detect the mouse beta 2-AR transcript. As shown in Fig. 1B, significant levels of the human beta 2-AR transcript were expressed only in the transgenic lines established from founders 2.2 and 4.3.

We next examined the distribution of transgene expression within the lung by performing in situ hybridization with the human beta 2-AR probe template that was used for the RPAs. Figure 2A shows that binding was present in distinct regions of the lung parenchyma of SP-C-beta 2-AR mice. Grains were found exclusively in clumps at the corners of alveoli, consistent with the known distribution of ATII cells. Specific hybridization was not detected in lung sections from nontransgenic mice (Fig. 2B) nor was it detected in SP-C-beta 2-AR sections hybridized with a riboprobe synthesized from the sense strand (Fig. 2C).


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Fig. 2.   Cellular distribution of SP-C-beta 2-AR transgene expression within the lung. In situ hybridization was performed on frozen lung sections with the human-specific beta 2-AR riboprobe. Dark-field views of sections counterstained with hematoxylin and eosin are shown. A: peripheral lung section from a SP-C-beta 2-AR transgenic mouse hybridized with the antisense riboprobe. The hybridization signal (arrows) is present in clusters located at the corners of alveoli, consistent with the distribution of alveolar type II (ATII) cells. No signal was detected in frozen lung sections from NTG mice hybridized with the antisense probe (B) or in SP-C-beta 2-AR sections hybridized with a sense riboprobe (C).

beta 2-AR expression and adenylyl cyclase activity. Having confirmed that the SP-C-beta 2-AR transgene was expressed in the lung and that the pattern of mRNA expression was consistent with the location of ATII cells, we next quantified beta 2-AR density by radioligand binding with [125I]iodocyanopindolol. For these experiments, binding was performed on membranes prepared from whole lung homogenates and is expressed as femtomoles of receptor per milligram of membrane protein. As shown in Fig. 3A, beta 2-AR density in membranes from nontransgenic mice was 194 ± 18 fmol/mg (n = 5). Receptor density in the transgenic lines with undetectable transcripts was not different from that in the nontransgenic lines (data not shown). In contrast, beta 2-AR density in mice from line 2.2, which had the highest level of transcript expression, was nearly fivefold greater than that in the nontransgenic mice (939 ± 113 fmol/mg; P < 0.001; n = 5). Transgenic mice from line 4.3, which expressed lower levels of transcript, displayed a smaller but still significant increase in beta 2-AR density compared with the nontransgenic group (349 ± 41 fmol/mg protein; P < 0.01; n = 4). Subsequent studies were carried out with the higher-expressing line 2.2.


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Fig. 3.   beta 2-AR expression and adenylyl cyclase activity. A: beta -AR density in membranes prepared from whole lung tissue was determined by radioligand binding with [125I]iodocyanopindolol (125ICYP). Nonspecific binding was determined in the presence of 10 µM propranolol. TG+, transgenic. Values are means ± SE from 4 or 5 independent experiments/group. B: adenylyl cyclase activity in NTG (open circle ) and SP-C-beta 2-AR () mice was determined with the same membranes prepared for the radioligand binding assays. The reactions were incubated with the indicated concentrations of isoproterenol ([isoproterenol]) for 10 min at 37°C. Values are means ± SE from 3 different experiments/group. No difference between NTG and SP-C-beta 2-AR mice was detected.

It has been suggested that the lung parenchyma may contain a population of "spare" beta 2-ARs (1). If so, it is possible that an increase in receptor density in such cells in the SP-C-beta 2-AR mice may not significantly increase maximal agonist-stimulated adenylyl cyclase activity compared with that in nontransgenic mice. Nevertheless, a decrease in the EC50 for the agonist (i.e., leftward shift of the dose-response curve) is realized under such circumstances. To determine whether receptor-mediated adenylyl cyclase activity was enhanced in SP-C-beta 2-AR mice, we measured adenylyl cyclase activity in the lung membranes used for the radioligand binding studies. Figure 3B shows that the basal adenylyl cyclase activities in nontransgenic and SP-C-beta 2-AR mice were not different (39.5 ± 3.68 and 36.7 ± 5.92 pmol · min-1 · mg-1, respectively). We also found no difference in maximal agonist-stimulated activity (61.4 ± 3.45 and 57.9 ± 8.62 pmol · min-1 · mg-1, respectively). However, the EC50 for isoproterenol in the SP-C-beta 2-AR mice was lower than that in the nontransgenic mice (60.0 ± 9.1 and 132.7 ± 35.4 nM, respectively; P < 0.05).

AFC studies. To assess the in vivo effects of beta 2-AR overexpression and activation in ATII cells, we measured AFC rates in nontransgenic and SP-C-beta 2-AR mice using an in situ lung preparation (9). As in a prior study by Ma et al. (19), AFC was calculated from the increase in the concentration of an impermeant volume indicator (131I-albumin) that occurred over 15 min after instillation into the airspace. Figure 4A shows that the basal AFC rate in SP-C-beta 2-AR mice was 38% greater than that in the nontransgenic mice (10.9 ± 0.6 and 15.0 ± 1.4%; P < 0.005). To assess whether increased AFC in the transgenic mice was the result of an increase in the pool of spontaneously activated receptors or an increased sensitivity to endogenous catecholamines, mice in both groups were adrenalectomized. Adrenalectomy had no effect on basal AFC in nontransgenic mice (11.6 ± 0.9%). In contrast, basal AFC in the SP-C-beta 2-AR mice was significantly reduced by adrenalectomy (to 9.7 ± 0.2%), which was no different from AFC in nontransgenic mice. Enhancement of AFC in SP-C-beta 2-AR mice was therefore ameliorated by elimination of endogenous catecholamines.


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Fig. 4.   Alveolar fluid clearance (AFC) studies in NTG and SP-C-beta 2-AR mice. Lungs were instilled in situ with an isosmolar solution containing 131I-albumin as a volume marker. Values are means ± SE of AFC expressed as the percentage of fluid absorption over 15 min. A: basal AFC in the SP-C-beta 2-AR mice (n = 7) was 38% greater than that in the NTG mice (* P < 0.005; n = 17). Adrenalectomy (ADX; +) had no effect on NTG mice but significantly reduced AFC in the SP-C-beta 2-AR mice compared with non-ADX (-) TG mice (Dagger  P < 0.01). B: measurements of AFC in NTG and SP-C-beta 2-AR mice were made after addition of the beta 2-agonist formoterol to the instillate. Both NTG and SP-C-beta 2-AR mice responded to formoterol in a dose-dependent fashion. AFC values for each dose tended to be higher in the TG mice but were significant only at baseline (* P > 0.005) and the 10-9 M dose (# P < 0.05).

We next examined whether in vivo agonist responsiveness was affected by ATII cell beta 2-AR overexpression. We used formoterol for these studies because it is a near full agonist that is beta 2-AR selective (42). Figure 4B shows that formoterol increased AFC in both nontransgenic and SP-C-beta 2-AR mice in a dose-dependent manner. AFC in the SP-C-beta 2-AR mice tended to be higher than that in the nontransgenic mice for each dose of formoterol studied, but the differences were not significant except for the lowest dose (10-9 M).

Na+-K+-ATPase alpha 1- and alpha 2-isoform expression. Previous studies (4, 26, 38) have suggested that beta -agonists may increase Na+ transport, and thus AFC, by increasing the activity of Na+-K+-ATPase. This effect may result from increased production or translocation of Na+-K+-ATPase subunits into the basolateral membrane, in particular the alpha 1-subunit isoform (4). This finding, together with the observations that 1) the alpha -subunit contains the catalytic region of Na+-K+-ATPase (18) and 2) the alpha 1-isoform is also the most abundant alpha -isoform in the lung (29), prompted us to examine whether expression of the alpha 1-isoform was altered in the lungs of SP-C-beta 2-AR mice. Using Western blot analysis with a monoclonal antibody specific for the alpha 1-isoform, we found no difference in alpha 1-isoform content in microsomal membranes prepared from the lungs of nontransgenic and SP-C-beta 2-AR mice (Fig. 5).


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Fig. 5.   Expression of Na+-K+-ATPase alpha -subunit isoforms in the mouse lung. A: Western blots of microsomal proteins prepared from pooled lungs were probed with the alpha 1-specific monoclonal antibody alpha 6f or the alpha 2-specific monoclonal antibody McB2. Microsomes prepared from skeletal muscle (sk.m.) were included as a positive control. B: densitometry of blots containing 60 µg of microsomal protein showed a 75% increase in the level of alpha 2-isoform expression but only a minimal change in alpha 1-isoform expression.

Although the alpha 1-isoform of Na+-K+-ATPase predominates in the lung, the alpha 2-isoform is present as well (29). Recently, James et al. (11) showed that a reduction in alpha 2-isoform expression in knockout mice was associated with significant isoform-specific changes in heart contractility (11). We considered, then, that the expression of the alpha 2-isoform might be different in the SP-C-beta 2-AR transgenic mouse. For these studies, Western blot analysis was performed on microsomal preparations from whole lung homogenates with the previously characterized McB2 monoclonal antibody. As shown in Fig. 5, alpha 2-isoform expression in the lungs from SP-C-beta 2-AR mice was ~75% greater than that in lungs from nontransgenic mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Agonists have been shown to stimulate AFC in a number of different mammalian species, including studies of excised human lungs (2, 3, 12, 34, 35). It has also been shown that endogenous catecholamines increase AFC in animal models of pulmonary edema caused by neurological insult (16), hemorrhagic shock (30), and sepsis (31). These catecholamine-mediated increases in AFC were blocked by the beta -antagonist propranolol, indicating that the beta -AR signaling system may have an important protective function that serves to prevent or limit alveolar flooding and enhance its resolution. Activation of the beta 2-AR signaling cascade by agonists or other means could therefore be a potential treatment for pulmonary edema as previously suggested (40).

Recent studies have demonstrated that receptor overexpression can be a useful method of activating beta 2-AR signaling pathways in a cell type-specific manner. For example, McGraw et al. (24) found that the contractile response to methacholine was markedly inhibited in transgenic mice that overexpressed the beta 2-AR in airway smooth muscle. In fact, these transgenic mice were less reactive to methacholine than nontransgenic mice treated with a beta -agonist. Agonist-independent enhancement of cardiac function has also been observed in transgenic mice that overexpressed the beta 2-AR in cardiomyocytes (25, 39). These studies show that the activation of receptor signaling by overexpression can effectively mimic agonist-mediated activation. This overexpression strategy can further be used to distinguish the physiological effects of beta 2-AR activation among different cell types within a complex tissue or organ by using cell-specific promoters to target expression. In addition, because desensitization does not appear to be appreciable in these overexpressing mice, genetic transfer may be superior to continuous high-dose agonist where tachyphylaxis could limit effectiveness.

A primary objective of this study was therefore to determine whether AFC could be persistently upregulated by overexpression of the beta 2-AR in ATII cells. Using the rat SP-C promoter to direct expression to ATII cells, we generated transgenic mice that expressed the beta 2-AR approximately fourfold over that of their nontransgenic littermates. Physiological assessment of these SP-C-beta 2-AR mice showed that their basal AFC rate was ~40% greater than that of the nontransgenic mice and was equivalent to AFC rates in nontransgenic mice treated with the beta -agonist formoterol. The SP-C-beta 2-AR mice also displayed a trend toward a further increase in AFC when they were treated with formoterol, but the difference did not reach significance. Nevertheless, these results show that overexpression of the beta 2-AR is as effective as short-term beta -agonist treatment with regard to enhancing AFC in vivo.

The cell types responsible for beta 2-AR-mediated increases in AFC have not been clearly elucidated in prior work, primarily because agonists could not be delivered in a cell-specific manner. A key role for the ATII cell has been supported by histological evidence that suggests that the ATII cell has the metabolic and anatomic potential to support the active transepithelial fluxes required for driving fluid transport. Studies of isolated ATII cells have also demonstrated the presence of basolateral Na+-K+-ATPase, apical Na+ channels, and functional beta 2-ARs (reviewed in Ref. 20). However, ATI cells may also have beta 2-ARs on their surface (5), and there is evidence to suggest that these cells have Na+-K+-ATPase activity as well (33). Activation of beta 2-ARs on other cells lining the alveolus, such as vascular smooth muscle and possibly vascular endothelium (6), could potentially alter vascular capacity and additionally contribute to the effects of beta -agonists on AFC (10). By selectively targeting expression (and thus receptor activation) to the ATII cell, we were able to eliminate the potential confounding effects of beta 2-AR activation in other cell types. With this strategy, we found that the increase in AFC due to transgenic activation of beta 2-AR signaling in ATII cells alone was nearly the same as that of nontransgenic mice treated with formoterol, an agonist that presumably acted on all cell types lining the alveolus. The absence of any additional increase in AFC by activation of receptors on these other cell types suggests that the effects of beta -agonists on AFC are mediated predominantly through their effects on ATII cells. Still, because the ATII cell is the progenitor of the ATI cell, it is possible that overexpression of the beta 2-AR is being maintained after differentiation. However, previous work (36) has shown that the half-life of receptor turnover in vivo is ~18 h, and in situ hybridization studies with many transgenes directed by the SP-C promoter, including those of the present work, showed that transgene expression is limited to ATII cells (14). It is therefore unlikely that persistent expression of the beta 2-AR transgene in ATI cells occurred in the absence of ongoing transcriptional activity.

Different modeling techniques indicate that the increase in basal (i.e., agonist-independent) receptor signaling that occurs with beta 2-AR overexpression may be due to a larger pool of spontaneously active receptors (24, 25). However, in an in vivo setting, the physiological effect of beta 2-AR overexpression could be the result of enhanced sensitivity to endogenous agonists (i.e., epinephrine). In measurements of isolated lung membrane adenylyl cyclase activity, we observed a leftward shift in the dose-response curve for agonist-stimulated adenylyl cyclase activity in the transgenic mice but no increase in maximal agonist activity. An increase in tachyphylaxis or desensitization is unlikely to explain this finding because overexpression tends to attenuate desensitization by increasing the number of receptors available for interacting with agonists. Moreover, an increase in sensitivity without a change in the maximal response is predicted by the current models in the case of receptor overexpression, to the point where some other component becomes the limiting factor in the maximal achievable response (i.e., the spare receptor phenomenon) (1). Our results thus support the concept that spare beta 2-ARs are present in the peripheral lung.

To further confirm that heightened agonist sensitivity was the mechanism underlying the in vivo gain of function in the SP-C-beta 2-AR mice, we performed adrenalectomies to minimize the effects of endogenous catecholamines (5). Mice were studied within 12 h of surgery to minimize the non-catecholamine-mediated effects of adrenalectomy on AFC, such as those resulting from the loss of corticosteroid production. Although adrenalectomy had no effect on the basal AFC rate in nontransgenic mice, AFC rates in the SP-C-beta 2-AR mice were significantly reduced compared with those in the nontransgenic mice. The finding of an effect only in the beta 2-AR overexpressors is further consistent with catecholamine depletion as the primary mechanism of adrenalectomy in this study. Taken together, these observations indicate that the increased AFC rate in SP-C-beta 2-AR mice was indeed the result of heightened sensitivity to endogenous catecholamines.

Having demonstrated that AFC could indeed be increased by beta 2-AR overexpression in ATII cells, we began to explore the cellular mechanisms by which it occurred. Experiments performed on cultured ATII cells have shown that beta -agonists increase Na+-K+-ATPase activity. Na+-K+-ATPase is an integral membrane protein localized to the basolateral membrane surface of ATII cells that transports three sodium ions out of the cell and two potassium ions into the cell for each molecule of ATP consumed (reviewed in Ref. 17). It is a heteromeric enzyme composed of a 97-kDa alpha -subunit and a 53-kDa glycosylated beta -subunit. The alpha -subunit contains both the cation and nucleotide binding sites and is the catalytic component of the enzyme. The beta -subunit is thought to regulate heterodimer assembly and stability, trafficking to the basolateral surface, and possibly the control of potassium ion kinetics. The mechanism by which beta 2-ARs regulate Na+-K+-ATPase function is not entirely clear. Previous work by Bertorello et al. (4) showed that isoproterenol increased the amount of the alpha 1-isoform, the predominant Na+-K+-ATPase alpha -isoform in the lung, in the basolateral membranes of cultured ATII cells. Because of the rapid onset of the effect, their interpretation was that beta -agonists increased Na+-K+-ATPase activity by recruiting alpha 1-subunits from an intracellular compartment to the basolateral surface. We therefore measured alpha 1-subunit expression in lung microsomes with Western blot analysis. Our results showed that alpha 1-subunit expression in SP-C-beta 2-AR mice was not different from that in the nontransgenic mice. This finding is analogous to that of Suzuki et al. (38), who found that the increase in Na+-K+-ATPase activity caused by terbutaline was not associated with changes in alpha 1-subunit expression. Our findings are, however, different from those of the former investigators (4). This difference could be partly due to our use of microsomal fractions derived from whole lungs rather than isolated ATII cells, which may have caused us to underestimate the changes that occurred specifically in these cells.

Despite these differences, recent data bring into question whether increases in alpha 1-subunit expression could account for beta 2-AR-mediated increases in AFC in vivo. Using adenoviral-mediated gene transfer, Factor et al. (7) found that in vivo delivery of the alpha 1-subunit to rat lungs did not result in an increase in AFC rates. In contrast, delivery of the beta 1-subunit increased both Na+-K+-ATPase activity and AFC rates. Because the beta 1-subunit does not possess catalytic activity, this effect may have resulted from increased formation and recruitment of active heterodimers. However, the absence of an effect by alpha 1-subunit overexpression could indicate that the increase in AFC due to beta 1-subunit overexpression resulted from interactions with other alpha -isoforms, although this possibility was not specifically addressed in their study (7). To date, four different Na+-K+ ATPase alpha -isoforms have been identified, with each having a unique tissue distribution (18). Transcripts for the alpha 2-isoform have been detected in RNA prepared from whole lung homogenates, but the abundance is less than that of the alpha 1-isoform (29). Nevertheless, even this small amount of alpha 2-isoform could have potential importance with regard to physiological function. The ratio of alpha 1- to alpha 2-isoform expression in the heart is similar to that in the lung (29), yet James et al. (11) showed that the alpha 2-isoform had a distinct role in regulating heart contractility in vivo by using knockout mice that expressed reduced levels of this isoform.

Given these observations, we considered the possibility that beta 2-AR activation in ATII cells could regulate AFC by modulating Na+-K+-ATPase alpha 2-subunit expression. Western blots of microsomal fractions prepared from whole lungs showed that alpha 2-isoform expression in the SP-C-beta 2-AR mice was ~75% greater than that in nontransgenic mice. Given that the beta 2-AR is not a secreted product and that in situ hybridization showed a pattern of transgene expression limited to ATII cells, the increase in alpha 2-isoform content that we observed was most likely the result of changes in ATII cell expression. In light of the findings by James et al. (11) demonstrating the physiological relevance of low-level alpha 2-subunit expression in the heart and the previously cited work by Factor et al. (7) showing that increased alpha 1-subunit expression does not increase AFC, our findings suggest the possibility that an additional mechanism of beta 2-AR-mediated increase in AFC may be due to regulation of other Na+-K+-ATPase alpha -subunit isoforms. However, activation of the beta 2-AR is necessary to elicit this increase in AFC because loss of circulating epinephrine from adrenalectomy ablated the effect. Of note, although an increase in the expression of the Na+-K+-ATPase alpha 2-subunit may play a role in beta 2-AR-mediated increases in AFC, we have not directly assessed this. Studies to differentiate alpha 2-subunit function by oubain sensitivity and direct overexpression of the alpha 2-subunit via transgenesis are necessary to confirm this link. The possibility that other components of ion transport, such as the apical Na+ channel or Cl- channel, are additionally upregulated in these mice must also be considered.

Of note, our data indicate that the increased basal AFC due to beta 2-AR overexpression in ATII cells likely results from enhanced signaling to endogenous catecholamines. This is supported by the leftward shift in agonist dose-response curves that was observed in the in vitro assays of adenylyl cyclase activity. We must also consider, though, that second messenger pathways other than cAMP may underlie beta 2-AR-mediated effects on AFC. Indeed, Suzuki et al. (38) observed that acute treatment of cultured ATII cells with beta -agonists produced increases in Na+-K+-ATPase that could not entirely be explained by changes in cAMP content. These findings, though, may have been related to the short duration of beta 2-AR activation because a later study (26) suggested a more prominent role for cAMP-dependent processes when beta -agonist exposure was prolonged (i.e., 5-7 days). However, it is of interest to note that tumor necrosis factor-alpha , which may actually lower cAMP, has been shown to increase AFC (32). Furthermore, a number of recent investigations have demonstrated that the beta 2-AR can couple to Ca2+-activated K+ channels (15), Cl- channels (8), and the Na+/H+ exchange regulator (37) independently of cAMP. Such coupling could explain the upregulation of basal AFC observed in the absence of increased adenylyl cyclase activity and cAMP levels.

In summary, we have used a cell-specific promoter to overexpress the beta 2-AR in ATII cells of transgenic mice, thereby permitting us to selectively delineate the effects of the ATII cell beta 2-AR from those of other cells in an in vivo setting. Our findings demonstrate that overexpression of the beta 2-AR in ATII cells can increase AFC as effectively as beta -agonist treatment. Transgenic activation of beta 2-AR signaling was associated with upregulation of the Na+-K+-ATPase alpha 2-isoform, suggesting, but not conclusively proving, a potential role for this isoform in regulating AFC. Last, these findings support the notion that activation of beta 2-AR signaling could be used as a therapeutic option to enhance the resolution of pulmonary edema.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the Caroline Halfter Spahn Trust Genetic Research Fund and National Heart, Lung, and Blood Institute Grants HL-03986, HL-41496, HL-55184, and HL-51856.


    FOOTNOTES

* Dennis W. McGraw, Norimasa Fukuda, and Paul F. James contributed equally to this work.

Address for reprint requests and other correspondence: S. B. Liggett, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0564 (E-mail: stephen.liggett{at}uc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 October 2000; accepted in final form 1 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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