We determined if prolonged isoproterenol (Iso) infusion in rats impaired the ability of the β2-adrenergic agonist terbutaline to increase alveolar liquid clearance (ALC). We infused rats with Iso (at rates of 4, 40, or 400 μg · kg−1 · h−1) or vehicle (0.001 N HCl) for 48 h using subcutaneously implanted miniosmotic pumps. After this time, the rats were anesthetized, and ALC was determined (by mass-balance after instillation of Ringer lactate containing albumin into the lungs) under baseline conditions and after terbutaline administration. Baseline and terbutaline-stimulated ALC in vehicle-infused rats averaged, respectively, 19.6 ± 1.2% (SE) and 44.7 ± 1.5%/h. The ability of terbutaline to increase ALC was eliminated at 400 μg · kg−1 · h−1 Iso, inhibited by 26% at 40 μg · kg−1 · h−1 Iso, and was not affected by 4 μg · kg−1 · h−1 Iso. β-adrenergic receptor (βAR) density of freshly isolated alveolar epithelial type II (ATII) cells from Iso-infused rats was reduced by the 40 and 400 μg · kg−1 · h−1 infusion rates. These data demonstrate that prolonged exposure to β-agonists can impair the ability of β2-agonists to stimulate ALC and produce ATII cell βAR downregulation.
- lung fluid balance
- pulmonary edema
- β-adrenergic receptor
- receptor downregulation
- alveolar epithelial type II cell
in severe pulmonary edema, fluid may flood the alveolar spaces and impair the oxygenation of the blood. During recovery from pulmonary edema, removal of this excess alveolar fluid is critical for the restoration of normal O2 transport. Recent studies have provided evidence of how this problem is solved by showing that excess water is cleared from the alveoli by a mechanism involving active ion transport across the alveolar epithelium (26, 40). Specifically, sodium enters the alveolar epithelial type II (ATII) cell through multiple specialized pathways located in the apical membrane and is then pumped out at the basolateral membrane by the Na+-K+-ATPase (2, 4, 7, 8, 16, 20, 26,33, 40). Water follows passively from the air spaces, possibly via specific water-only pathways (aquaporins) (44). The lung alveolar epithelium thus plays an active and important role in removing excess liquid from its air spaces during resolution of severe pulmonary edema.
It is now well accepted that administration of β-adrenergic agonists significantly increases the rate of alveolar transepithelial sodium and, consequently, water transport in most species, including humans (4, 6-8, 11, 16, 20, 25, 38, 39, 46). Additionally, endogenous epinephrine release during development and different pathological conditions has been shown to acutely increase the rate of liquid clearance from the air spaces (13, 23, 36). These observations suggest that β-agonists might be used clinically to accelerate the recovery of pulmonary edema in patients (2,15) and that recovery from some forms of edema may in fact be naturally accelerated by endogenous epinephrine (23, 36). Although this is a provocative hypothesis, the long-term effectiveness of either β-agonist therapy or endogenous epinephrine stimulation is not clear because of the potential for β-adrenergic receptor (βAR) desensitization (a regulated process in which continued agonist exposure to its receptor attenuates the receptor's biological effect) and downregulation (a form of desensitization in which receptor number decreases) (3, 31). Thus if ATII cell βARs undergo a significant degree of desensitization, the efficacy of β-agonist therapy or of endogenous epinephrine would diminish over time. Consequently, the major objective of this study was to determine if a 48-h infusion of a broad range of isoproterenol (Iso) doses in rats impaired the ability of terbutaline, a β2-agonist, to increase alveolar liquid clearance (ALC) and, if so, whether the impairment was accompanied by a downregulation in ATII cell βAR number.
A total of 113 male Sprague-Dawley rats weighing 250–300 g (Harlan Sprague Dawley, Chicago, IL) was used in this study. The rats were housed in the Animal Care Facility at the Northeastern Ohio Universities College of Medicine for at least 1 wk under temperature-controlled conditions [20 ± 2 (SD)°C] and at a relative humidity of 50 ± 10% before experimental use. The rats were fed a standard rat chow and had water ad libitum. All rat experiments were approved by the Northeastern Ohio Universities College of Medicine Institutional Animal Care and Use Committee.
48-h Iso Infusions
Miniosmotic pumps (Alzet model 2001, Durect, Cupertino, CA) were filled under sterile conditions with either the βAR agonist (−)-isoproterenol (+)-bitartrate (Iso; Sigma Chemical, St. Louis, MO) or its vehicle (0.001 N HCl) and primed in sterile saline at 37°C overnight. The next morning, the filled pumps were aseptically implanted subcutaneously in the dorsum superior to the midthorax of rats under halothane anesthesia. Iso concentrations were selected to allow the pumps to deliver the drug at infusion rates ranging between 4 and 400 μg Iso base · kg−1 · h−1. These Iso administration rates were selected, because they represent the range of infusion rates that have been used in previous studies to evaluate the ability of Iso to produce βAR downregulation (18, 42,43).
Determination of ALC
ALC was measured as previously described (6). The rats were anesthetized with 80 mg/kg ip pentobarbital sodium (Abbot Laboratories, N. Chicago, IL), with anesthetic being supplemented as needed. The body temperature was monitored using a rectal temperature probe and was maintained with a water-perfused heating pad. A polyethylene tracheal cannula (PE-240, Clay Adams, Becton-Dickinson, Sparks, MD) was placed in the rat's airway via a tracheotomy and connected to a mechanical ventilator (Harvard Apparatus, Nantucket, MA). The lungs were ventilated with 100% O2 at a respiratory rate of 40 breaths/min with an average tidal volume of 2.7 ± 0.1 (SD) ml. Peak inspiratory pressure was 9.5 ± 1.3 Torr under baseline conditions, and end-expiratory pressure was atmospheric. The rat was placed at a 45° angle (head elevated), and a polyethylene catheter (PE-50, Clay Adams) was inserted through a port in the tracheal cannula and into the lungs for liquid instillation. The rats were allowed to stabilize 10 min after surgery before the start of the experiment. At this time, 3 ml/kg of a 5% bovine serum albumin (BSA, Sigma) solution in Ringer lactate (Baxter Healthcare, Deerfield, IL) was instilled into the left lung at a rate of 0.20 ml/1.5 min using a 1-ml syringe. The solution was previously adjusted with NaCl to an osmolality of 315 mM. The alveolar instillate was left in the lungs for 1 h beginning at the completion of instillation. At this time, a thoracotomy was done, and a blood sample was obtained for blood gas analysis via aortic puncture and analyzed using a Radiometer system. Average blood gas determinations were: Po 2, 315 ± 139 (SD) Torr; Pco 2, 34.1 ± 8.3 Torr; pH, 7.42 ± 0.07. The rat was then killed by exsanguination, the lungs were removed, and the remaining instilled liquid was aspirated for analysis of albumin concentration by refractometry (American Optical, Buffalo, NY). The refractometer was calibrated by a series of albumin standards (Sigma). ALC was determined using the following mass balance equation: ALC = (1 − Albi/Albf)(100), where ALC is expressed as the percentage of the instilled liquid that left the air spaces during the 1-h observation period, and Albi and Albf are the initial and final albumin concentrations of the instillate, respectively.
Quantification of ATII cell βAR density
ATII cell isolation.
The ATII cells were isolated by the technique of Dobbs et al. (9). Briefly, the rats were anesthetized with pentobarbital sodium (80 mg/kg ip) and heparinized (1,000 U). A tracheotomy was done, and an 18-gauge angiocatheter (Becton Dickinson Infusion Therapy Systems, Sandy, UT) was inserted and tied in place in the trachea. The chest was opened, and the lungs were removed. The lungs were perfused through the pulmonary artery to remove blood and lavaged to remove alveolar macrophages. The lungs were digested with 20 ml elastase (3 U/ml, Worthington Biochemical, Lakewood, NJ) for 20 min at 37°C. The digested tissue was minced in the presence of fetal bovine serum (Hyclone, Logan, UT) and DNase (Sigma) and filtered through sterile gauze and then through 70-μm nylon mesh filters (Becton Dickinson Labware, Franklin Lakes, NJ). The filtrate was centrifuged at 913 g, and the cell pellet resuspended in Dulbecco's modified Eagle medium (DMEM, Irvine Scientific, Santa Ana, CA) containing an antibiotic-antimycotic cocktail of penicillin G, streptomycin sulfate, and amphotericin B (GIBCO BRL, Grand Island, NY) and glutamine (Irvine Scientific). The type II cells were purified by differential adherence to IgG (Sigma)-coated plates. Cell purity (94%) was determined by using a Beckman Coulter Z1 Coulter particle counter, and yield was determined on a hemocytometer. A sufficient number of ATII cells could be isolated from the lungs of each rat to allow a complete receptor binding analysis for each animal. βAR binding assays were done immediately on the freshly isolated ATII cells as described below.
The total number of cellular β-adrenergic receptor sites (cell membrane bound and internalized receptors) was determined using the lipophilic radioligand [125I]-iodocyanopindolol (ICYP; New England Nuclear, Life Science Products, Pittsburgh, PA), as described by Fabisiak et al. (12). Briefly, the isolated cells were homogenized with a Polytron for 10–15 s in 20 ml of 50 mM ice-cold Tris buffer (pH 7.4) containing 10 mM MgCl2. The homogenate was centrifuged for 10 min at 40,000 g. The resulting cell pellet was resuspended in 20 ml Tris buffer (pH 7.4) containing 1 mM EDTA (Sigma) and incubated for 30 min at 36°C to dissociate away endogenous ligand or competing drug from the receptor binding site. The EDTA wash procedure was repeated a second time. This wash protocol has been found to effectively remove bound agonists from βARs in ATII cells (5). Binding assays in a total volume of 0.5 ml were conducted in polystyrene assay tubes containing homogenized membranes of ∼1 × 105 cells/tube and the following concentrations of [125I]-ICYP: 300, 200, 100, 75, 50, 25, 10, and 5 pM. Nonspecific binding of [125I]-ICYP was defined by the presence of 2 μM (s)-(−)-propranolol (Sigma). The homogenates were incubated for 60 min at 36°C. The assays were terminated by rapid filtration through Whatman GF/B filters previously soaked in 0.05% polyethylenimine (Sigma) using a Brandel R48 filtering manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed three times with 7 ml ice-cold Tris buffer and then counted in a gamma counter. βAR density (Bmax) and the equilibrium dissociation constant (K d) were determined using the LIGAND curve-fitting program (NIH). Bmax determinations were normalized by expressing the data as a fraction of the tissue protein concentration (determined by the Bio-Rad protein assay).
Effect of prolonged Iso infusion on ability of β2AR agonists to increase ALC.
We initially determined whether a prolonged Iso infusion to rats impaired the ability of the β2AR agonist terbutaline to increase ALC by implanting miniosmotic pumps containing Iso in concentrations calculated to produce Iso infusion rates of 4, 40, or 400 μg · kg−1 · h−1. After 48 h, the rats were anesthetized with pentobarbital sodium, and ALC was measured under either baseline conditions or after stimulation with 10−4 M terbutaline (Sigma) added to the instillate. For each Iso infusion rate, ALC was also determined in separate vehicle-infused rats either under baseline conditions or after terbutaline instillation. Thus for each Iso infusion rate, four groups of rats (n = 6–7 in each group) were studied in a randomized fashion as depicted in Fig. 1.
Macromolecular permeability of the alveolocapillary barrier after Iso infusion.
The data obtained in this study indicated that the ability of instilled terbutaline to stimulate ALC was impaired in rats that had been infused with Iso at rates of 40 and 400 μg · kg−1 · h−1 (seeresults). To rule out the possibility that these observations could be explained by possible Iso-induced changes in the alveolocapillary barrier permeability, we measured the pulmonary vascular and alveolar epithelial permeability in rats infused with either Iso (400 μg · kg−1 · h−1,n = 3) or vehicle (n = 3). At 48 h after pump implantation, the rats were anesthetized with pentobarbital sodium and prepared as described in Determination of ALCwith the exception that a polyethylene catheter (PE-50, Clay Adams) was inserted into a carotid artery for injections and blood pressure and heart rate measurements. After a 10-min period to allow for stabilization of blood pressures and heart rate, 2 ml/kg of a solution containing 2.5 mg/ml of FITC-conjugated dextran 70,000 (FD70; Sigma) was injected intra-arterially. This dose was selected to give a blood concentration of ∼0.05 mg FD70/ml. The FD70 was run through a PD-10 column (Pierce Endogen, Rockford, IL) before the injection to separate free unbound FITC from the injected FD70. Arterial blood samples (0.8 ml) were obtained at 10 and 20 min after the FD70 injection. At 30 min into the study, 3 ml/kg of the albumin solution was instilled into the lungs and the rats were studied as described inDetermination of ALC for 1 h, with the exception that ALC was not determined. At the end of the 1-h experimental time period, another blood sample was taken, the rat was killed by exsanguination, and the lungs were removed through a thoracotomy. The lungs were homogenized, and FD70 fluorescence was measured in plasma, homogenate, and supernatant after centrifugation at 14,000 g. Hemoglobin and hematocrit were measured in the blood. The extravascular lung water content was determined according to the method of Selinger et al. (41).
The clearance of the vascular tracer FD70 across the lung endothelium into the lung extravascular compartments (endothelial leak) was measured spectrophotofluorometrically (Fluoroskan Ascent FL, Labsystems, Helsinki, Finland). FD70 passage across the endothelial-epithelial barriers was considered equal to that of albumin, because they have similar molecular masses (70 vs. 68 kDa). Total extravascular FD70 accumulation in the lung was calculated as the difference between the total lung FD70 (in lung homogenate and in the alveolar samples) and the vascular space FD70. Vascular space FD70 was calculated by multiplying the final plasma sample fluorescence by the calculated plasma volume in the lungs, as previously described (33). Extravascular FD70 accumulation in the lung was expressed as plasma equivalents, i.e., volume of extravasated plasma in milliliters that would account for the extravascular fluorescence in the lung. Transepithelial FD70 leak was determined from the ratio of alveolar to plasma FD70 fluorescence.
Effect of pulmonary hemodynamics on terbutaline-stimulated ALC after Iso infusion.
To determine whether possible Iso-induced changes in pulmonary hemodynamics could have influenced the ability of instilled terbutaline to increase ALC in Iso-infused rats, we determined ALC in additional rats infused with Iso (400 μg · kg−1 · h−1,n = 6) or vehicle (n = 6) for 48 h. Immediately before determining ALC, we killed these rats with an overdose of pentobarbital sodium and then measured ALC in the nonperfused lungs in the presence of 10−4 M terbutaline in the instillate. The alveolar epithelium of such nonperfused lung preparations has been previously shown to retain its ability to clear alveolar liquid for extended periods of time (16, 38). For these experiments, ALC was measured as described in Determination of ALC with the exceptions that the lungs were inflated at a constant pressure [6.7 ± 0.5 Torr (SD)] using a water overflow system with a heated and humidified gas mixture of 95% O2and 5% CO2, and the rat's internal temperature was monitored from a temperature probe placed adjacently to the diaphragm through a small incision in the abdominal cavity.
ATII cell βAR density after Iso infusion.
To determine whether the impaired terbutaline-stimulated ALC was accompanied by a reduction in the total ATII cell βAR number, we quantified βAR density on freshly isolated ATII cells obtained from rats receiving either Iso (at the rates of 4, 40, or 400 μg · kg−1 · h−1) or vehicle for 48 h (n = 4–7 for each infusion type). At this time, the rats were anesthetized with pentobarbital sodium, the lungs were removed, the ATII cells were isolated, and Bmaxand K d were determined as described inQuantification of ATII Cell βAR Density.
Multigroup comparisons of data were done by analysis of variance (ANOVA) followed by post hoc testing using the Student Newman-Keuls test. Paired and unpaired comparisons were made, respectively, by paired and unpaired Student's t-tests when appropriate. A preliminary analysis of the data indicated that there were no differences in ALC in the three groups of vehicle-infused rats in which ALC was determined under baseline conditions or in the three groups of vehicle-infused rats in which ALC was measured after terbutaline instillation. Accordingly, these data were averaged to provide a single vehicle infusion/baseline ALC group and a single vehicle/terbutaline stimulation group that was used for the statistical comparisons.
Systemic Effects of Iso Infusion
Rats infused with vehicle or Iso at 4 μg · kg−1 · h−1 exhibited, respectively, average 3.8 ± 0.3 (SE) % (P < 0.001) and 5.9 ± 0.5% (P < 0.001) weight gains during the 48-h period after pump implantation. These mean values were not significantly different. In contrast, rats infused with Iso at the rates of 40 and 400 μg · kg−1 · h−1 exhibited, respectively, no weight change and a 5.3 ± 0.7% weight loss (P < 0.001) during this 48-h period. The weight response observed in the latter two groups was significantly different (P < 0.05) from that observed in the vehicle-infused rats. Iso (400 μg · kg−1 · h−1)- and vehicle-infused rats exhibited no differences in arterial blood pressure, whereas the heart rate was significantly increased at this Iso infusion rate [vehicle: 360 ± 0, Iso: 500 ± 20 (SE) beats/min; P = 0.05]. Arterial blood gases were not altered by any Iso infusion rate, nor was there any evidence of edema in the noninstilled lung in Iso-infused rats.
Although we did not measure plasma Iso concentrations in this study, it was possible to estimate the steady-state plasma concentrations for the 40 and 400 μg · kg−1 · h−1infusion rates based on measurements made in three previous studies that have determined plasma Iso concentrations in rats infused with Iso at rates of 50, 80, 110, 150, and 400 μg · kg−1 · h−1 using subcutaneously implanted miniosmotic pumps (18, 42, 43). In analyzing the data from these studies, we found that there was a highly significant (P < 0.005, r = 0.99,r 2 = 0.97) correlation between the Iso infusion rate and the plasma Iso concentration. Based on the regression equation derived from this data [plasma Iso concentration (nM) = (0.1226)(infusion rate) − 0.4999], we estimated plasma Iso concentrations for the infusion rates of 40 and 400 μg · kg−1 · h−1 to be, respectively, 4.4 nM (0.9 ng/ml) and 48.5 nM (10.3 ng/ml).
Effects of Prolonged Iso Infusion on Ability of β2AR Agonists to Increase ALC
The average baseline ALC observed in all vehicle-infused rats (n = 18) was 19.6 ± 1.2 (SE) % of the instilled liquid absorbed per hour (Fig. 2). ALC in vehicle-infused rats given terbutaline in the instillate (n = 18) was 128% greater (44.7 ± 1.5%) than that observed in vehicle-infused rats in which ALC was determined under baseline conditions (Fig. 2).
Iso infusion at the 4 μg · kg−1 · h−1 infusion rate did not affect the ability of terbutaline to increase ALC (Fig.2). ALC after terbutaline instillation was significantly reduced (P < 0.05) in a dose-dependent fashion in rats infused with Iso at the 40 and 400 μg · kg−1 · h−1 rates (Fig.2). Baseline ALC was significantly increased (P < 0.05) in rats infused with Iso at 4 and 40 μg · kg−1 · h−1 but not at 400 μg · kg−1 · h−1.
Macromolecular permeability of the alveolocapillary barrier after iso infusion.
No differences in endothelial leak (as measured by extravascular plasma equivalents) and epithelial leak (as determined from the alveolar/plasma fluorescence ratio) were observed between Iso (400 μg · kg−1 · h−1) and vehicle-infused rats (Fig. 3). The extravascular lung water content (reflecting the sum of the baseline lung water volume and remaining alveolar instillate) was identical in both groups of rats (vehicle, 5.37 ± 0.17; Iso, 5.37 ± 0.12 g H2O/g blood-free dry lung wt).
Effect of Pulmonary Hemodynamics on Terbutaline-Stimulated ALC After Iso Infusion
Because there was a clear tachycardiac effect from the Iso infusion, we investigated the possibility that changes in pulmonary hemodynamics might have affected ALC. In rats without pulmonary blood flow, ALC was significantly lower (P < 0.001) in Iso-infused rats (400 μg · kg−1 · h−1) after terbutaline stimulation (18.3 ± 1.9%) than in the vehicle-infused rats (33.8 ± 1.6%).
ATII Cell βAR Density After Iso Infusion
Bmax did not differ in freshly isolated ATII cells obtained from rats infused with vehicle or Iso at a rate of 4 μg · kg−1 · h−1 but was significantly reduced (P < 0.05) in ATII cells harvested from rats infused with Iso at 40 and 400 μg · kg−1 · h−1 (Fig.4). K d averaged 28.2 ± 2.6 pM in the vehicle-infused rats and did not change with any infusion rate.
In this study, we found that 48 h of continuous Iso infusion in rats resulted in a dose-dependent impairment in the ability of an instilled β2-agonist, terbutaline to increase ALC (Fig.2). Additionally, the increase in ALC observed in rats infused with Iso was also reduced at the highest Iso infusion rate (Fig. 2). These data thus demonstrate that a prolonged β-agonist administration to intact animals can lead to a reduction in the ability of the alveolar epithelium to increase water transport in response to β2-agonist stimulation. Additionally, the observation that the βAR density of freshly isolated ATII cells from rats infused with Iso at rates of 40 and 400 μg · kg−1 · h−1 was reduced suggested that βAR downregulation might have played a role in mediating the impaired ability of terbutaline to increase ALC.
The ability of terbutaline to increase ALC appeared to be progressively reduced with each incremental increase in the Iso infusion rate (Fig.2). It is important, however, to interpret these data in relationship to the baseline ALC for each specific Iso infusion rate. In this regard, at the 4 μg · kg−1 · h−1 Iso infusion rate, ALC was increased 51.5% over that observed in vehicle-infused rats and could be further increased by terbutaline administration (Fig. 2). At the 40 μg · kg−1 · h−1 Iso infusion rate, ALC was elevated to a similar degree to that observed at 4 μg · kg−1 · h−1, but no further increase in ALC was observed after terbutaline administration. Finally, ALC was not increased at the 400 μg · kg−1 · h−1 Iso infusion rate and was refractory to terbutaline administration. Thus terbutaline did not increase ALC higher than the value produced by Iso alone at either the 40 or the 400 μg · kg−1 · h−1 Iso infusion rate.
Although our study was not specifically designed to address the question of whether the Iso infusion impaired the ability of this β-agonist (Iso) to increase ALC, the data were suggestive of such an effect. In this regard, ALC was significantly lower in rats infused with Iso at 400 μg · kg−1 · h−1 compared with the elevated ALCs observed at the two lower Iso infusion rates (Fig. 2). Additionally, the observation that the ALC was no higher in rats infused with Iso at 40 μg · kg−1 · h−1 compared with that observed in those receiving the 4 μg · kg−1 · h−1 Iso infusion rate is suggestive of an impaired response. This possibility is further supported by the observation that ALC could not be further increased by terbutaline instillation in the rats infused with Iso at 40 μg · kg−1 · h−1 (Fig.2). A definitive answer to this question would, however, require a study design incorporating a comparison of ALC in rats infused with Iso at a time before desensitization occurred.
Although a significant increase (51.5%) in ALC was observed in rats infused with Iso at 4 μg · kg−1 · h−1 (Fig. 2), it is important to note that this magnitude of stimulation is not the maximal possible by the alveolar epithelium of the intact rat lung. In this regard, acute terbutaline (10−4 M) instillation in this study produced a much larger increase in ALC (128%), and Saldı́as et al. (39) have reported that ALC increased by ∼150% when high Iso concentrations (10−5M) were delivered acutely via the perfusate of an isolated perfused rat lung preparation during a 1-h study period. In preliminary experiments, we observed similar increases in ALC to that observed by Saldı́as et al. (39) when Iso was added to the alveolar instillate at a concentration of 10−5 M (data not shown). Although these observations clearly indicate that it is possible to acutely produce large increases in ALC by exposing the lung to high concentrations (10−4–10−5 M) of β-agonists, the prolonged administration of such doses is likely to result in a downregulated ability of the alveolar epithelium to respond to β-agonist therapy over time, because the infusion of Iso at rates (40 and 400 μg · kg−1 · h−1) calculated to produce much lower plasma Iso concentrations (4.4 and 48.5 nM) did not increase ALC to values higher than that observed at an Iso infusion rate of 4 μg · kg−1 · h−1.
Prolonged Iso infusion in rats has previously been shown to result in a number of significant systemic cardiovascular and metabolic alterations. For example, Hayes et al. (18) observed significant weight loss, cardiac hypertrophy, and a diminished maximal rate of left ventricular pressure development in rats infused with Iso at a rate of 400 μg · kg−1 · h−1 for 4 days. In this study, we also observed that rats infused Iso for 48 h at this rate lost weight and exhibited an elevated heart rate. Although the impaired ability of terbutaline to stimulate ALC in the Iso-infused animals most likely resulted from a desensitization event involving alveolar epithelial β2ARs, the development of nonspecific Iso-induced systemic effects raised the possibility of other explanations for our ALC results. Accordingly, we evaluated the possibility that the observed impairment in terbutaline-stimulated ALC might have either a hemodynamic or an increased alveolocapillary protein permeability basis. To rule out the possibility of a pulmonary hemodynamic explanation, we compared the ability of instilled terbutaline to increase ALC in both Iso (400 μg · kg−1 · h−1)- and vehicle-infused rats under conditions of no pulmonary blood flow. We found that terbutaline increased ALC in the vehicle-infused rats but not in the Iso-infused animals. These results were analogous to those observed in live rats and thus indicate that the impaired ability of terbutaline to stimulate ALC could not be explained by potential differences in pulmonary hemodynamics. To determine if an Iso-induced increase in the alveolocapillary protein permeability could have caused ALC to be underestimated (because of an increased leakage of the albumin tracer from the air spaces), we measured vascular endothelial and alveolar epithelial permeability in Iso (400 μg · kg−1 · h−1)- and vehicle-infused rats. No differences in the permeability of either barrier were observed in either of the two groups (Fig. 3), thus indicating that the impairment in the ability of terbutaline to stimulate ALC in the Iso-infused rats was not the result of increased protein permeability. Finally, the observation that the extravascular lung water was identical in Iso- and vehicle-infused rats further strengthened the evidence that pulmonary hemodynamics and/or increased protein permeability did not play significant roles in the inhibition of the ability of terbutaline to increase ALC in Iso-infused rats.
How do the Iso infusion rates evaluated in this study relate to those that have been used therapeutically? Iso has been intravenously infused in relatively low doses to treat cardiac dysrhythmias and in higher doses to treat reactive airway diseases (34, 37, 45). For example, Reyes et al. (37) reported that pediatric intensive care patients received intravenous Iso infusions at average rates of 1.7 ± 0.1 μg · kg−1 · h−1 for cardiac patients and 30.0 ± 12.6 μg · kg−1 · h−1 for patients with pulmonary diseases. (One patient received Iso at a rate of 330 μg · kg−1 · h−1.) Although the authors do not state the total duration of treatment, they report that they obtained blood samples for plasma Iso concentration an average 54.4 ± 9.5 h (range: 1.5–240 h) after an effective steady-state infusion rate was achieved. These data indicate that the Iso infusion rates and duration of treatment that have been used clinically fall within the range of those evaluated in our study and, further, raise the possibility that the impairment in the β2-agonist-stimulated ALC observed in our study (particularly at the 40 μg · kg−1 · h−1 infusion rate) might have relevance to humans treated with this drug. Although more specific β2-agonists (e.g., albuterol) are now more commonly used in clinical practice, there is evidence that chronic administration of therapeutically relevant concentrations of these drugs also produces desensitization phenomena in some types of lung cells. In this regard, Kelsen et al. (21) found that freshly isolated airway epithelial cells from normal individuals who received inhaled albuterol (180 μg four times daily for 7 days) exhibited a reduced βAR density, a reduced cAMP production in response to Iso administration, and an increased βAR kinase (βARK, an enzyme responsible for phosphorylating the receptor and producing desensitization) protein immunoreactivity.
Is there a correlation between the number of βARs at the alveolar level in the lungs and the observed decrease in ALC after prolonged Iso infusion? The prolonged administration of β-agonists is well known to result in βAR downregulation in many cell types (3, 31); however, there is little information available about βAR regulation in the distal alveolar epithelium. Therefore, we measured βAR density in freshly isolated ATII cells from rats infused with Iso and found that the Iso infusion decreased Bmax to a similar degree in rats infused with Iso at 40 and 400 μg · kg−1 · h−1 but had no effect on Bmax at the 4 μg · kg−1 · h−1 Iso infusion rate (Fig. 4). These observations thus suggest that ATII cell β2AR downregulation might have played a role in producing the observed impairment in terbutaline-stimulated ALC and that the phenomenon of βAR downregulation is more widespread in the lung than previously thought. There is evidence to suggest, however, that other desensitization mechanisms must have played a role in producing the dose-dependent ALC responses. In this regard, although both the 40 and 400 μg · kg−1 · h−1infusion rates resulted in similar reductions in Bmax, ALC was lower in rats infused with Iso at 400 than at 40 μg · kg−1 · h−1 regardless of whether ALC was measured under baseline conditions or after terbutaline instillation (Fig. 2).
βAR desensitization has been shown to occur via an orchestrated set of mechanisms that have effective time courses varying from minutes to days. Short-term (occurring within minutes to hours) mechanisms involve phosphorylation of the βAR and, in turn, uncoupling from the stimulatory G proteins (24, 35). Long-term regulation occurs over periods of hours or days and involves internalization/degradation of βARs (1) and inhibition of gene expression and transcription (17). In addition to these desensitization events occurring at the level of the receptor, there is also evidence that desensitization can occur at points further downstream in the βAR signal transduction cascade. In this regard, McMartin and Summers (29) observed that Iso infusion in rats (14 days at 400 μg · kg−1 · h−1) produced impairments in cardiac tissue βAR signaling at sites downstream of the receptor occurring between the receptor and adenylyl cyclase and at the level of the cAMP-dependent protein kinase. Additionally, studies evaluating the mechanisms of βAR desensitization in lung membranes obtained from rats after chronic β-agonist exposure have identified impairments in βAR signaling that include βAR downregulation, impaired β-agonist-stimulated cAMP production, altered βAR-Gs coupling, and increased phosphodiesterase activity (14, 30, 32). Finally, it is possible that ion transport protein (e.g., Na+ channel, Na+-K+-ATPase) activity and/or expression might be affected (10). There are thus numerous sites in the alveolar epithelial βAR signaling pathway and ion transport machinery that could potentially be affected by prolonged β-agonist administration.
Although the mechanisms of βAR desensitization have been evaluated in a number of isolated lung cell types (e.g., airway epithelial cells, alveolar macrophages, and airway smooth muscle cells) (21, 22,28), it is difficult to extrapolate these results to the ATII cell because of the possibility of cell type-specific responses (21, 28). For example, McGraw and Liggett (28) observed that human airway smooth muscle cells exhibited very little functional desensitization in response to prolonged β2AR stimulation compared with that observed in mast cells and that this difference was related to the heterogeneity in the expression of βARK. Additionally, there is evidence that human airway epithelial and alveolar macrophage βARs exhibit different sensitivities to desensitization (21). Our identification of a reduced βAR density in freshly isolated ATII cells from rats infused with Iso indicates that the ATII cell βAR is capable of undergoing at least one of the putative mechanisms of desensitization. This observation is consistent with that of a preliminary study in which we found that exposure of isolated rat ATII cells to Iso (10−6 M) for 48 h significantly reduced the βAR density (5). The current data extend these in vitro observations by demonstrating that reductions in ATII cell βAR density can occur in vivo when the intact animal is exposed to much lower circulating Iso concentrations (4.4 to 48.5 nM). However, the extent to which other βAR desensitization mechanisms occur in the ATII cell remains to be investigated.
Given the ability of β2-agonists to acutely increase alveolar epithelial sodium and water transport (4, 6-8, 11,16, 20, 25, 38, 39, 46), it has been suggested that β-agonists might be used clinically to accelerate the recovery of pulmonary edema in patients (2, 15). Although the mechanisms responsible for desensitization of the ATII cell βAR remain to be fully elucidated, the results of this and previous studies begin to suggest how β-agonist therapy might be potentially used for the treatment of pulmonary edema. First, because many studies have demonstrated that it is possible to produce significant increases in ALC for hours after stimulation with high β2-agonist concentrations (4,6, 16, 23, 25), it is likely that short-term (i.e., receptor phosphorylation) mechanisms of desensitization may play at most a minimal role in decreasing the ability of β-agonists to increase alveolar epithelial sodium and water transport. This conclusion is consistent with our previous results in which we observed that a 4-h intravenous epinephrine infusion (181 ng · kg−1 · min−1) in anesthetized rats did not impair the ability of this catecholamine to increase ALC (6) and suggests that high dose β-agonist therapy could produce significant increases in ALC in the short-term setting. The results of the current study suggest, however, that prolonged administration of β-agonists at high doses will eventually make the alveolar epithelium refractory to β2-agonist stimulation. The observation that ALC was increased by 51.5% by the 4 μg · kg−1 · h−1 Iso infusion rate without producing functional and receptor downregulation suggests, however, that β2-agonist therapy in relatively low doses might be effective in producing clinically useful increases in ALC over an extended time period. It is interesting to note, however, that ALC measured under baseline conditions was not different in rats infused with Iso at rates of either 4 or 40 μg · kg−1 · h−1 (Fig. 2), even though receptor number was reduced at the 40 μg · kg−1 · h−1 Iso infusion rate (Fig. 4). These data thus suggest that even in a receptor-downregulated state (as produced by the 40 μg · kg−1 · h−1 Iso infusion rate) clinically useful increases in ALC may still be achieved. Other approaches, such as attempting to increase ATII cell β2AR numbers by gene manipulation (10, 27) or modifying the structure of the β2-agonist to make it less likely to induce βAR downregulation (19) might also prove useful.
In conclusion, we found that a 48-h Iso infusion to intact rats impaired the ability of β2-agonists to increase ALC in a dose-dependent manner. To our knowledge, this is the first study showing desensitization of the β2-agonist-stimulated ALC response and downregulation of ATII cell βARs after prolonged β-agonist exposure of animals. The observation of a downregulated ATII cell βAR population suggests that a decreased receptor number might play a role in producing the impaired β2-agonist ALC stimulation, but the potential involvement of other βAR signaling pathway and ion channel protein defects needs to be investigated.
The authors thank Dr. David Jarjoura for advice with the statistical analysis.
This study was supported by American Heart Association, Ohio Valley Affiliate Grant 0051029B; National Heart, Lung, and Blood Institute Grant HL-31070; and a grant from the Ohio Board of Regents Research Challenge program.
Address for reprint requests and other correspondence: M. B. Maron, Dept. of Physiology, Northeastern Ohio Univs. College of Medicine, 4209 State Rte. 44, P.O. Box 95, Rootstown, OH 44272-0095 (E-mail:).
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.
- Copyright © 2002 the American Physiological Society