Department of Physiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
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ABSTRACT |
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We determined if prolonged
isoproterenol (Iso) infusion in rats impaired the ability of the
2-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.
-adrenergic receptor (
AR) 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
-agonists
can impair the ability of
2-agonists to stimulate ALC
and produce ATII cell
AR downregulation.
lung fluid balance; pulmonary edema; -adrenergic receptor; receptor downregulation; alveolar epithelial type II cell
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INTRODUCTION |
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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 -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
-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
-agonist therapy or endogenous epinephrine stimulation is
not clear because of the potential for
-adrenergic receptor (
AR)
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
ARs undergo a
significant degree of desensitization, the efficacy of
-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
2-agonist, to
increase alveolar liquid clearance (ALC) and, if so, whether the
impairment was accompanied by a downregulation in ATII cell
AR number.
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METHODS |
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Animals
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 theDetermination 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: PO2, 315 ± 139 (SD) Torr; PCO2, 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 = (1Quantification of ATII cell AR 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. AR binding
assays were done immediately on the freshly isolated ATII cells as
described below.
[125I]-iodocyanopindolol binding.
The total number of cellular -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
ARs 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.
AR
density (Bmax) and the equilibrium dissociation constant
(Kd) 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).
Experimental Design
Effect of prolonged Iso infusion on ability of 2AR
agonists to increase ALC.
We initially determined whether a prolonged Iso infusion to rats
impaired the ability of the
2AR 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.
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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 · kg1 · h
1 (see
RESULTS). 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 ALC
with 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 in
Determination 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).
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 · kg1 · 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% O2
and 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 AR density after Iso infusion.
To determine whether the impaired terbutaline-stimulated ALC was
accompanied by a reduction in the total ATII cell
AR number, we
quantified
AR 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 Bmax
and Kd were determined as described in
Quantification of ATII Cell
AR Density.
Statistical analysis. 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.
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RESULTS |
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Systemic Effects of Iso Infusion
Rats infused with vehicle or Iso at 4 µg · kgAlthough 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 · kg1 · h
1
infusion 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, r2 = 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 2AR
Agonists to Increase ALC
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Iso infusion at the 4 µg · kg1 · 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 · kg1 · 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).
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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 · kgATII Cell AR Density After Iso Infusion
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DISCUSSION |
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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 2-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
-agonist administration to intact
animals can lead to a reduction in the ability of the alveolar
epithelium to increase water transport in response to
2-agonist stimulation. Additionally, the observation
that the
AR 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
AR 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 · kg1 · 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
-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 · kg1 · 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
Saldías et al. (39) have reported that ALC increased by ~150% when high Iso concentrations (10
5
M) 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
Saldías 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
-agonists, the prolonged administration of such doses is likely to
result in a downregulated ability of the alveolar epithelium to respond
to
-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 · kg1 · 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
2ARs, 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 · kg1 · 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
2-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
2-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
AR density, a reduced cAMP production in
response to Iso administration, and an increased
AR kinase (
ARK,
an enzyme responsible for phosphorylating the receptor and producing
desensitization) protein immunoreactivity.
Is there a correlation between the number of ARs at the alveolar
level in the lungs and the observed decrease in ALC after prolonged Iso
infusion? The prolonged administration of
-agonists is well known to
result in
AR downregulation in many cell types (3, 31);
however, there is little information available about
AR regulation
in the distal alveolar epithelium. Therefore, we measured
AR 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
2AR downregulation might have played a role in producing the observed impairment in terbutaline-stimulated ALC and that the
phenomenon of
AR 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
1
infusion 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).
AR 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
AR 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
ARs (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
AR 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
AR 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
AR desensitization in lung membranes
obtained from rats after chronic
-agonist exposure have identified
impairments in
AR signaling that include
AR downregulation,
impaired
-agonist-stimulated cAMP production, altered
AR-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
AR signaling pathway and ion transport machinery
that could potentially be affected by prolonged
-agonist administration.
Although the mechanisms of AR 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
2AR stimulation compared with that observed in
mast cells and that this difference was related to the heterogeneity in
the expression of
ARK. Additionally, there is evidence that human
airway epithelial and alveolar macrophage
ARs exhibit different
sensitivities to desensitization (21). Our identification
of a reduced
AR density in freshly isolated ATII cells from rats
infused with Iso indicates that the ATII cell
AR 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
AR
density (5). The current data extend these in vitro
observations by demonstrating that reductions in ATII cell
AR
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
AR desensitization mechanisms occur in the
ATII cell remains to be investigated.
Given the ability of 2-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
-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
AR remain to be fully
elucidated, the results of this and previous studies begin to suggest
how
-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
2-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
-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
-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
-agonists at high doses will eventually
make the alveolar epithelium refractory to
2-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
2-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
2AR numbers by gene manipulation (10, 27)
or modifying the structure of the
2-agonist to make it
less likely to induce
AR downregulation (19) might also
prove useful.
In conclusion, we found that a 48-h Iso infusion to intact rats
impaired the ability of 2-agonists to increase ALC in a
dose-dependent manner. To our knowledge, this is the first study
showing desensitization of the
2-agonist-stimulated ALC
response and downregulation of ATII cell
ARs after prolonged
-agonist exposure of animals. The observation of a downregulated
ATII cell
AR population suggests that a decreased receptor number
might play a role in producing the impaired
2-agonist
ALC stimulation, but the potential involvement of other
AR signaling
pathway and ion channel protein defects needs to be investigated.
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ACKNOWLEDGEMENTS |
---|
The authors thank Dr. David Jarjoura for advice with the statistical analysis.
![]() |
FOOTNOTES |
---|
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: mbm{at}neoucom.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.
10.1152/ajplung.00381.2001
Received 25 September 2001; accepted in final form 6 December 2001.
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