Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, The University of Alabama at Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
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Active
Na+ transport by alveolar
epithelial cells has been demonstrated to contribute significantly to
alveolar fluid clearance. However, the contribution of transepithelial
Cl movement to the
reabsorption of isosmotic fluid across the alveolar epithelium in vivo
has not been elucidated. We hypothesized that Cl
transport could be
increased across the alveolar epithelium in vivo and across cultured
alveolar type II cells by agents that increase intracellular cAMP
(e.g., forskolin). In studies where 5% albumin in
sodium methanesulfonate (a
Cl
-free solution) was
administered into the lung, forskolin administration significantly
increased intracellular influx of
Cl
and fluid into the
alveolar space. In vitro studies with cultured rabbit alveolar type II
cell monolayers in Ussing chambers demonstrated that elevations in
intracellular cAMP increase short-circuit current by
increasing both Cl
secretion and Na+ reabsorption.
The cystic fibrosis transmembrane conductance regulator channel blocker
glibenclamide and the loop diuretic bumetanide partially decreased the
forskolin-induced increase in short-circuit current. These data may
explain the failure of agonist that stimulated intracellular cAMP to
increase alveolar fluid clearance in the rabbit. Moreover, the data
suggest that in the event Na+
absorptive pathways are damaged, transepithelial
Cl
secretion and the
consequent intra-alveolar fluid influx may be upregulated.
amiloride; sodium transport; terbutaline; short-circuit current; alveolar type II cells; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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THE PRIMARY FUNCTION of the lung is gas exchange, which occurs by simple diffusion. The presence of fluid in the alveolar space increases the diffusion distances for O2 and CO2 and results in ventilation-perfusion mismatch, arterial hypoxemia, and hypercapnia. Thus the efficient removal of fluid (transudate or exudate) across the normal and injured alveolar epithelium is of primary importance for the maintenance of normal lung function. Active transport of Na+ across the alveolar epithelium creates an osmotic force, which results in the reabsorption of alveolar fluid isotonic to plasma (14). A study (13) across isolated alveolar type II (ATII) cells indicated that Na+ diffuses passively across the ATII cell apical membranes and down a favorable electrochemical gradient maintained by Na+-K+-ATPase, and then is actively transported across the basolateral membranes of these cells by ouabain-sensitive Na+-K+-ATPase. Various studies in humans and animals indicated that active Na+ reabsorption plays an important role in diminishing alveolar fluid and improving oxygenation in both patients with cardiogenic and noncardiogenic edema (15) and animals with hyperoxic lung injury (18, 27).
The demonstration that alveolar fluid clearance (AFC) in vivo and ex vivo is upregulated by agents that increase the concentration of intracellular cAMP (3, 6, 9, 21) has led to speculation that these agents may be useful in limiting alveolar edema in a variety of pathological conditions. Patch-clamp studies of freshly isolated ATII cells and a putative Na+-channel protein isolated from ATII cells and reconstituted in lipid bilayers indicated that agents that increase cAMP increase the open probability of amiloride-sensitive Na+ single channels without affecting their conductance (22, 28). Furthermore, cAMP increased the activity of ATII cell Na+-K+-ATPase by an Na+-independent mechanism (26).
It has been thought that Cl
transport across alveolar epithelial cells occurred passively through
the paracellular junctions, driven by the existing electrochemical
forces. However, recent evidence indicates that cultured rat ATII cells
contain a cAMP-stimulated Cl
conductance in their
apical membranes (11). Measurements of short-circuit currents
(Isc) and
Cl
fluxes across monolayers
of rat ATII cells mounted in Ussing chambers were consistent with
transient increases in transepithelial Cl
reabsorption by agents
that increase cAMP (11, 12). However, the contribution of the
transepithelial Cl
movement
to the reabsorption of isosmotic fluid across the alveolar epithelium
of intact animals has not been elucidated. It is conceivable that in
some situations, especially when transepithelial
Na+ reabsorption has been
compromised by reactive species (10, 16), adult alveolar cells may
secrete Cl
,
which may cause a net influx of fluid into the alveolar
space. Fetal distal lung epithelial cells secrete
Cl
into the alveolar space
in utero, which subsequently drives fluid secretion (17).
We thus performed a series of experiments to study the reabsorption of
isotonic fluid instilled into the alveolar spaces of anesthetized
rabbits. To investigate the contribution of
Na+ and
Cl in this process, we
performed measurements with 1)
normal saline in the presence and absence of amiloride, which blocked
the entry of Na+ across
amiloride-sensitive Na+ channels,
and 2)
Cl
-free solutions. These
measurements were then repeated after intratracheal instillation of
forskolin, an agent known to increase cAMP levels in ATII cells (19).
We performed our experiments in rabbits because these animals due to
their size are better suited than rats for sequential measurements of
alveolar fluid. To elucidate the putative mechanisms, we also isolated
ATII cells from the lungs of rabbits, cultured them until they formed
tight monolayers, and then measured
Isc before and
after stimulation with forskolin or permeant analogs of cAMP. The
results of these studies indicate that transcellular movement of both
Na+ and
Cl
contribute to the
magnitude and direction of fluid fluxes across the alveolar epithelium
after cAMP stimulation.
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METHODS |
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The study was approved by the Animal Review Committee of the University of Alabama at Birmingham. All animals received humane care in compliance with the Principles of Laboratory Care formulated by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals prepared by the National Research Council (Washington, DC: Natl. Acad. Press, 1996).
Measurements of AFC in rabbits. Male
New Zealand White rabbits (Myrtle's Rabbits, Thompson Station, TN)
weighing 1.6-2.6 kg were allowed free access to food and water
before experimentation. All rabbits were anesthetized with an
intravenous injection of ketamine (10 mg/kg; Parke-Davis, Morris
Plains, NJ) via a marginal ear vein. Anesthesia was maintained
throughout the experimental procedure by the administration of inhaled
1% isoflurane (Abbott Laboratories, North Chicago, IL) carried in 99%
O2. After a tracheotomy, a
3.5-mm-ID endotracheal tube and a modified
4F Fogarty catheter (American Edwards Laboratory, Irvine,
CA) were placed into the trachea. The Fogarty catheter was modified by
removal of the balloon tip and placement of a yellow suture bootie
(Oxboro-Medical International, Ham Lake, MN) that was punctured on its
distal end. Placement of this catheter ~9-10 cm into the trachea
resulted in an airtight intubation of the right caudal lobe of the lung
that was confirmed postmortem. Rabbits were ventilated with a Harvard
Apparatus ventilator (model 683, Harvard Apparatus, Millis, MA). The
tidal volume and rate were adjusted to yield a peak inspiratory
pressure between 14 and 17 mmHg measured from within the endotracheal
tube and an arterial PCO2
(PaCO2) of 32-45
Torr. Pancuronium bromide (0.3 mg · kg1 · h
1;
Elkins-Sinn, Cherry Hill, NJ) was administered
intravenously to ensure relaxed chest wall muscle tone. Arterial
pressure was monitored by placement of a 22-gauge
central ear arterial catheter. All pressures were recorded on a Grass
model 7D polygraph (Grass Instruments, Quincy, MA). All rabbits
received a maintenance infusion of lactated Ringer at 4 ml · kg
1 · h
1,
and esophageal temperatures were maintained at 38-39°C with a
heating pad. A 15-min equilibration period followed completion of the
surgical preparation.
After equilibration, 10 ml of 5% fat-free bovine serum albumin (Sigma) suspended in solutions with varying ionic compositions (see below) were instilled into the right caudal lobe over a 2-min period. The dead space in the modified Fogarty catheter was cleared by injection of 600 µl of 100% O2. The fluid in the right caudal lobe was subsequently withdrawn in 1-ml increments 1 and 2 h after instillation, a 300-µl aliquot of the bronchoalveolar lavage fluid (BALF) was removed for analysis, and the remaining fluid was reinstilled in the right caudal lobe. The 300-µl samples were centrifuged at 1,000 g for 10 min to pellet cells and debris, and the protein concentration was determined by modification of a spectrophotometric method (24).
AFC expressed as a percentage of total instilled volume (excluding the
volume of albumin) was calculated from the following relationship, as
described previously (7, 27): AFC = (1 Ci/Ct)/0.95,
where the variables
Ci and
Ct are,
respectively, the protein concentrations at time
0 and at either 1 or 2 h. Time
0 was considered to be the end of the instillation. The
osmolality of the instillate and the BALF samples was determined with a
vapor pressure osmometer (model 5520, Wescor, Logan, UT). The pH,
PaCO2, arterial
PO2 (PaO2), and
HCO
3 of arterial blood samples were determined after 15 min of equilibration and every 30 min thereafter throughout the experimental period. All blood gas samples were analyzed
at 37°C with a blood gas analyzer (model 1306, Instrumentation Laboratory, Lexington, MA).
Four groups of rabbits (n = 12/group) were used to determine the effects of intratracheal cAMP instillation on AFC and its dependence on Na+ reabsorption. In the control group, 10 ml of 154 mM NaCl containing 5% BSA (NaCl-BSA) were instilled into the right lower lobe as described above. To investigate the contribution of Na+ reabsorption to AFC, we instilled NaCl-BSA containing 1 mM amiloride into a second group of rabbits. The concentration of amiloride in the instilled volume was determined by measuring the fluorescence intensity of alveolar samples at an excitation of 360 nm and emission of 415 nm (8). A third group of rabbits was instilled with NaCl-BSA containing 50 µM forskolin, and the fourth group was instilled with NaCl-BSA containing 50 µM forskolin and 1 mM amiloride.
The contribution of transepithelial
Cl transport in AFC was
determined by instilling a solution in which NaCl was replaced with
equimolar (154 mM) sodium methanesulfonate (pH
6.9-7.1, 301-304 mosmol).
Cl
concentrations of the
instillate and BALF samples were determined with a
Cl
analyzer (model 925, Ciba Corning Diagnostics, Midfield, MA). To evaluate the effects of
increased epithelial cAMP on AFC and Cl
influx into the alveolar
space, a second group of rabbits (n = 8) was instilled with a similar
Cl
-free sodium
methanesulfonate solution containing 50 µM forskolin. All rabbits
were euthanized with an overdose of pentobarbital sodium (65 mg/kg; The
Butler Company, Columbus, OH) after the 120-min instillate sample was obtained.
ATII cell isolation and culture. ATII cells were isolated from the lungs of young adult male New Zealand White rabbits (1.8-2.2 kg) as previously described (1). Briefly, the rabbits were anesthetized and killed by an intravenous pentobarbital sodium injection (100 mg/kg body wt). The lungs were ventilated with air, perfused with ~200 ml of cold phosphate-buffered saline, removed en bloc from the thoracic cavity, and lavaged with a balanced salt solution and buffered saline alternately to remove macrophages. Subsequently, the lungs were filled with Joklik's modified minimum essential medium (JMEM; 37°C) containing elastase (2 U/ml in JMEM; Worthington, Lakewood, NJ) and DNase. Proteolytic digestion was stopped after 30 min by the addition of cold (4°C) JMEM containing 10% heat-inactivated fetal bovine serum (FBS) and trypsin inhibitor (40 mg, soybean I-S; Sigma). The lungs were minced with dissecting scissors into small pieces, resuspended in the medium, and stirred for 20 min at 4°C. The suspension was filtered through nylon gauze of decreasing porosity (Tetko, Briarcliff Manor, NY) to remove debris. ATII cells were separated out of the crude cell suspension by discontinuous density-gradient centrifugation with Percoll (Sigma), and cell counts were determined with a hemocytometer. More than 85% of these cells were identified as ATII cells by the modified Papanicolaou stain or by a positive staining for alkaline phosphatase. Viability, quantified by trypan blue exclusion, was higher than 95%.
ATII cells were subsequently seeded onto 0.4-µm-pore, 0.33-cm2 Transwell tissue culture filters (Costar, Cambridge, MA) at a density of 1.5 × 106 cells/cm2 and maintained in JMEM containing 10% FBS, 0.1 µM dexamethasone, 100 µg/ml of penicillin, and 100 µg/ml of streptomycin at 37°C with 95% air-5% CO2. The medium was changed every other day after seeding.
Measurement of bioelectrical properties of ATII
cells. Spontaneous potential difference (PD) and
transepithelial resistance (Rt)
across ATII cell monolayers were measured daily, beginning after 2 days
in culture, with an Epithelial Voltohmeter equipped with
chopstick-style electrodes (World Precision Instruments, Sarasota, FL).
Only monolayers achieving
Rt values >800
· cm2 within
4-6 days after seeding were used in our studies.
Confluent ATII cell monolayers were mounted into modified Ussing
chambers, and their active ion transport properties were evaluated. The
monolayers were bathed in a solution containing (in mM) 145 Na+, 5 K+, 125 Cl, 1.2 Ca2+, 1.2 Mg2+, 25 HCO
3, 3.3 H2PO
4, and 0.8 HPO2
4 (pH 7.4) at 37°C gassed with
95% O2-5% CO2 (solution
1). Glucose (10 mM) and mannitol (10 mM) were added to the basolateral and apical solutions, respectively. We substituted glucose with mannitol in the apical solution to minimize the
contribution of a putative sodium-glucose cotransporter
to Na+ influx into ATII cells. A
transepithelial voltage clamp (EC-825, Warner Instruments, Hamden, CT)
was used for the continuous monitoring of
Isc. After ATII
cell monolayers were mounted in the Ussing chambers, the
Isc was allowed
to stabilize before each experiment was begun (~10 min). The
Rt was determined
with Ohm's law from the observed change in
Isc resulting
from 5-s square-voltage pulses (1 or 2 mV) imposed
across the monolayer.
To test the hypotheses mentioned previously, different combinations of 1) 10 µM amiloride, 2) 10 µM forskolin or 100 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), 3) 200 µM glibenclamide, or 4) 100 µM bumetanide were added to the medium. Amiloride and bumetanide were applied to the apical and basolateral sides of the cell monolayers, respectively, whereas the other compounds were applied to both sides of the monolayer.
Statistical analysis. All variables are expressed as means ± SE. Analysis of the effect of cAMP on transepithelial ion transport (in vivo) and AFC was conducted with one-way ANOVA. The Student-Newman-Keuls test was utilized for post hoc comparisons. Analysis of the effects of the different pharmacological interventions on heart rate, arterial blood pressure, arterial blood gas parameters, and peak inspiratory pressure was conducted with ANOVA. In vitro data were analyzed with an unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Effects of forskolin on AFC in the presence of NaCl. Forskolin administration resulted in a small increase in AFC at 60 and 120 min postinstillation compared with the control group, but this difference did not reach significance (Fig. 1). Rabbits administered amiloride had significantly decreased AFC compared with the control group at 60 and 120 min postinstillation (P < 0.05). Similarly, animals administered amiloride and forskolin had significantly decreased AFC compared with animals in the forskolin group at 60 and 120 min postinstillation (P < 0.05). At 60 min postinstillation, animals administered amiloride and forskolin had a significantly greater AFC than those administered amiloride (P < 0.05). These results indicate that the small increase in AFC after instillation of forskolin was due to reabsorption of Na+ through non-amiloride-inhibitable pathways. Figure 1B depicts the amiloride-sensitive fraction of AFC, which is the difference in group mean AFC values between the control and amiloride (and forskolin and forskolin with amiloride) groups.
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There was no significant difference in instillate amiloride
concentration between the two groups administered amiloride. The values
for the animals administered amiloride alone were 0.77 ± 0.01 and
0.68 ± 0.03 mM at 60 and 120 min postinstillation, respectively.
The values for the animals administered amiloride and forskolin were
0.78 ± 0.01 and 0.66 ± 0.03 mM at 60 and 120 min
postinstillation, respectively. Multiple regression analysis was
performed, with the model including time, the presence or absence of
forskolin, and amiloride concentration in the instilled alveolar fluid
as independent variables and AFC as the dependent variable. The
multiple regression coefficient was 0.64 (P < 0.0001), with amiloride
concentration and the presence of forskolin significantly influencing
AFC. Time did not significantly contribute to AFC in this model. The
data are consequently depicted as the measured values of AFC vs. the
concentration of amiloride in the instilled fluid in the presence or
absence of forskolin (Fig.
2). There was a negative
association between AFC and amiloride concentration in the instilled
fluid in the presence (r = 0.52; P < 0.01) or absence
(r =
0.62;
P < 0.01) of forskolin.
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Effects of forskolin on AFC in the presence of
Cl-free solutions.
Rabbits administered sodium methanesulfonate were observed to have a
significantly decreased AFC compared with the control group (i.e.,
rabbits that were instilled with NaCl) at 60 and 120 min
postinstillation (P < 0.05; Fig.
3). Rabbits instilled with sodium methanesulfonate and forskolin demonstrated a negative AFC
(i.e., fluid secretion into the alveolar space) at 60 min postinstillation and essentially zero AFC at 120 min. However, there
was no significant difference in AFC between the two sodium methanesulfonate groups at 120 min postinstillation. Forskolin administration resulted in a significantly greater alveolar instillate Cl
concentration 60 min
after instillation compared with animals administered sodium
methanesulfonate alone (Fig.
3B). However, there were no
significant differences in instillate osmolality between the groups at either time point (Fig.
3C).
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DISCUSSION |
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The bioelectric properties of the rabbit ATII cell monolayers in the present study are in good agreement with those in rats (4, 5, 10, 11). The fact that the Isc was decreased to ~20% of its control value after instillation of 10 µM amiloride into the apical bath (Fig. 4B) indicates that Na+ entered the apical membranes of rabbit ATII cells via amiloride-sensitive ion channels. This conclusion is further supported by our previous whole cell patch-clamp findings indicating the presence of amiloride-sensitive currents in rabbit ATII cells (12).
Immediately after the application of either forskolin or cAMP to both
sides of the monolayers, there was a sharp increase in the
Isc (Fig. 4). The
fact that a similar increase in the
Isc was observed
after forskolin application in the presence of amiloride strongly
implies that these agents stimulated
Cl secretion across the
ATII cell monolayers. The rapid increase in the
Isc likely
reflects the efflux of Cl
from the cells across their apical membrane. The subsequent decrease would be expected as intracellular
Cl
moves toward its
equilibrium concentration. However, the elevated steady-state
Isc after
forskolin instillation in the presence of amiloride reflects the
sustainable level of Cl
secretion by these cells. The sensitivity of the forskolin-induced Isc to both
bumetanide and glibenclamide suggests that
Cl
is loaded into these
cells, at least in part, via a
Na+-K+-2Cl
cotransporter in the basolateral membrane and exits across the apical
membrane through the CFTR
Cl
-channel protein.
The role of Cl in the cAMP
response of rat ATII cells has been previously elucidated. In the study
by Cheek et al. (5), an increase in intracellular cAMP resulted in a
transient decrease in the
Isc, followed by
a sustained increase. The resulting steady-state Isc was partially
inhibited by amiloride, suggesting that the cAMP response was due to
increased Na+ absorption. However,
in a subsequent study (12) from this group, isotopic flux measurements
revealed that both Na+ and
Cl
absorption were
increased after cAMP. The results of Jiang et al. (11) indicated that
treatment of rat ATII cells with terbutaline stimulated apical membrane
Cl
channels, with
functional and pharmacological properties similar to CFTR. They also
concluded that Cl
influx
into the cells couples with Na+
influx to increase net NaCl absorption without affecting the apical
membrane Na+ conductance. Despite
the qualitative differences among the aforementioned studies and ours,
taken as a whole, the data indicate that agents that increase
intracellular cAMP in ATII cells induce transepithelial Cl
fluxes.
Yue et al. (28) previously showed that agents that increase cAMP also
increase the open probability and mean open time of Na+ channels in freshly isolated
rat ATII cells. Furthermore, similar results were noted when protein
kinase A was added along with ATP to the cytoplasmic site of inside-out
patches of rat ATII cells (28) as well as to the
trans (cytoplasmic) site of lipid bilayers containing an Na+-channel
protein isolated from rabbit ATII cells (22). Protein kinase A also
phosphorylated the rabbit ATII cell
Na+-channel protein (2), a finding
that has been previously associated with activation of
Na+ channels (25). The significant
increase in amiloride-sensitive Na+
Isc after
elevation of intracellular cAMP is consistent with these previous data
and suggest that cAMP increases
Na+ absorption in this species.
The failure of forskolin or terbutaline to increase AFC is likely due
to the complexity of the cAMP response in stimulating both
Cl secretion and
Na+ absorption. On the basis of
these data, we anticipated that cAMP would have increased
transepithelial Na+ absorption
across rabbit ATII cell monolayers. Although, the mean
amiloride-sensitive
Isc after
forskolin was increased over control cell monolayers, the differences
were not significant. Taken in total, these in vitro data indicate that
the cAMP-induced transport responses in rabbit ATII cells are complex
and involve both Na+ and
Cl
transport pathways.
Our in vivo measurements of AFC offer additional insight into the
coupling of Na+ and
Cl movement across the
alveolar epithelium. In the presence of normal saline, forskolin
increased AFC by ~20%. However, this change was not significant and
was brought about by a large increase in the amiloride-insensitive
fraction of AFC. These data contrast with measurements of
Isc in which
forskolin resulted in a sustained increase in
Isc. It is
possible that this difference is due to nonspecific phenotypic changes
in cultured ATII cells. More likely, it may simply reflect the higher
sensitivity of
Isc measurements where each filter was used as its own control.
Our data further demonstrate that removal of
Cl from the instillate
reduces the AFC from 15 to ~0%. It is possible that the decrease in
AFC may be due to the differences in size of
Cl
(35 Da) and methanesulfonate (95 Da)
molecules. However, this is unlikely because forskolin induced a net
Cl
secretion into the
alveolar space as shown by the doubling of the intra-alveolar
Cl
concentration at 60 min,
resulting in significant fluid influx into the alveolar space. In the
presence of Cl
,
Na+ reabsorption resumed as shown
by the fact that AFC returned to zero at 120 min. Finally, the
osmolality of the instilled alveolar fluid did not change
significantly, indicating isotonic fluid reabsorption due to the uptake
of both Na+ and
Cl
.
One factor that could have confounded our in vivo AFC measurements was the administration of isoflurane, a halogenated anesthetic. Rats exposed to either isoflurane or halothane for 6 h were observed to have a decreased AFC compared with control animals (20). However, rats exposed to isoflurane for 2 h did not exhibit a significantly decreased AFC (20). Furthermore, the coadministration of 98% O2 or terbutaline abrogated the halothane-mediated decrease in AFC (20). In the present study, rabbits were coadministered 99% O2 with 1% isoflurane for ~2.5 h. Finally, the amiloride-sensitive fraction of AFC in the present study is similar to that reported by Smedira et al. (23). Consequently, although isoflurane administration may have influenced our observations, the short period of experimentation and the coadministration of 99% O2 likely decreased most isoflurane-mediated effects on AFC.
The teleological implications of our findings are potentially relevant
to clinical scenarios of acute lung injury. The ability to clear
alveolar fluid has been associated with improved morbidity in the
setting of acute respiratory distress syndrome (15). The
process of AFC may involve a balance between active
Na+ absorption and
Cl secretion, and
perturbations in active Na+
absorption by reactive
O2-N2
intermediates (10, 16) may result in relatively unopposed
Cl
secretion and resultant
alveolar flooding. Indeed, if the concurrent pathophysiology includes a
shock state (e.g., sepsis, hemorrhage), Cl
secretion may be
upregulated by increased circulating catecholamines, further
exacerbating alveolar fluid accumulation. The experiments involving
sodium methanesulfonate would support the possibility of a clinical
scenario of increased alveolar flooding after epithelial cAMP is
increased via
2-adrenoreceptor agonism.
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ACKNOWLEDGEMENTS |
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This project was supported by National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173 and Office of Naval Research Grant N00014-97-1-0309. V. G. Nielsen is the recipient of American Heart Association (Southeast Affiliate) Grant-In-Aid 9810091SE. M. D. DuVall is a Parker B. Francis Fellow.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: V. G. Nielsen, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., JT 845E, Birmingham, AL 35233-1924.
Received 11 June 1998; accepted in final form 9 September 1998.
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