Division of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago, Illinois 60616; and Departamento de Enfermedades Respiratorias, Pontificia Universidad Católica de Chile, Santiago, Chile
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ABSTRACT |
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-Adrenergic agonists have been
reported to increase lung liquid clearance by stimulating active
Na+ transport across the alveolar
epithelium. We studied mechanisms by which
-adrenergic isoproterenol
(Iso) increases lung liquid clearance in isolated perfused fluid-filled
rat lungs. Iso perfused through the pulmonary circulation at
concentrations of 10
4 to
10
8 M increased lung liquid
clearance compared with that of control lungs
(P < 0.01). The increase in lung
liquid clearance was inhibited by the
-antagonist propranolol
(10
5 M), the
Na+-channel blocker amiloride
(10
4 M), and the antagonist
of Na-K-ATPase, ouabain (5 × 10
4 M). Colchicine, which
inhibits cell microtubular transport of ion-transporting proteins to
the plasma membrane, blocked the stimulatory effects of Iso on active
Na+ transport, whereas the isomer
lumicolchicine, which does not affect cell microtubular transport, did
not inhibit Na+ transport. In
parallel with these changes, the Na-K-ATPase
1-subunit protein abundance and
activity increased in alveolar type II cells stimulated by
10
6 M Iso. Colchicine
blocked the stimulatory effect of Iso and the recruitment of
Na-K-ATPase
1-protein to the
basolateral membrane of alveolar type II cells. Accordingly, Iso
increased active Na+ transport and
lung liquid clearance by stimulation of
-adrenergic receptors and
probably by upregulation of apical
Na+ channels and basolateral
Na-K-ATPase mechanisms. Recruitment from intracellular pools and
microtubular transport of Na+
pumps to the plasma membrane participate in
-adrenergic stimulation of lung liquid clearance in rat lungs.
active sodium transport; lung edema clearance; apical sodium channels; sodium-potassium-adenosinetriphosphatase; cytoskeleton
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INTRODUCTION |
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PULMONARY EDEMA ACCUMULATES as the result of changes in hydrostatic and colloid-osmotic pressure gradients or changes in the vascular or epithelial permeability (11, 19). Over the last decade, lung epithelial Na+ transport has been explored in physiological models of mammalian lungs (1, 10, 15, 29) and alveolar epithelial type II cell monolayers (6, 12, 13, 18, 33, 37). It has been shown that Na+ enters the alveolar epithelial cells through apical Na+ channels and is actively transported out of the cell by basolaterally located Na-K-ATPase (18, 24). Water probably moves along osmotic gradients generated by active Na+ transport out of the alveolus.
Studies in vivo and in isolated perfused fluid-filled lungs have
reported that -adrenergic agonists enhance active
Na+ transport and lung liquid
clearance in mammals (3, 7, 15, 30). In cultured alveolar type II
(ATII) cell monolayers, the intracellular second messenger cAMP
participates in the
-adrenergic stimulation (6, 13).
Administration of -adrenergic agonists terbutaline, epinephrine, and
isoproterenol (Iso) has been reported to increase active Na+ transport across the
alveolocapillary membrane of rat lungs (7, 15, 30). Recently, it has
been reported that incubation of rat ATII cells for 15 min with 1 µM
Iso increased 2.5-fold the Na-K-ATPase activity, and this effect was
abolished by a cAMP-dependent protein kinase inhibitor (27).
Intracellular microtubular transport and cortical cytoskeleton have
been shown as well to play a role in signal transduction and recruiting
of ion transport-related proteins to the plasma membrane (4, 5, 17,
26). The purpose of this study was to examine the mechanisms by which
Iso stimulates lung liquid clearance and focus on the contribution of
apical Na+ channels and
basolaterally located Na-K-ATPases. Subsequently, we examined whether
disruption of the cellular microtubular transport pathway of
ion-transporting proteins contributes to Iso-stimulated active
Na+ transport.
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MATERIALS AND METHODS |
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Pathogen-free male Sprague-Dawley rats weighing 280-360 g were
purchased from Harlan Sprague Dawley (Indianapolis, IN). A total of 140 rat lungs were studied. All animals were provided food and water ad
libitum and were maintained on a 12:12-h light-dark cycle. Iso,
amiloride, ouabain, propranolol, colchicine, and -lumicolchicine were purchased from Sigma (St. Louis, MO).
Isolated Lungs
The isolated lung preparation was performed as previously described (25, 29, 34). Briefly, rats were anesthetized with 50 mg/kg body wt of pentobarbital sodium. A tracheotomy was performed, and the rats were mechanically ventilated with a tidal volume of 2.5 ml, a peak airway pressure of 8-10 cmH2O, and 100% O2 for 5 min. During the thoracotomy, rats were anticoagulated with 400 U of heparin. The pulmonary artery and left atrium were cannulated, and the pulmonary circulation was flushed of remaining blood by perfusing with a buffered salt albumin solution containing 135.5 mM Na+, 119.1 mM ClPerfusion of the lungs was performed with 90 ml of the same buffered salt albumin solution containing 0.16 mg/ml of fluorescein-tagged albumin (FITC-albumin; Sigma). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump and from there flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary arterial pressures were maintained at 0 and 12 cmH2O, respectively, and were recorded via a pressure transducer with a zero reference point at the level of the left atrium. Pulmonary arterial pressure and left atrial pressure were recorded continuously with a multichannel recorder (Gould 3000 oscillograph recorder; Gould, Cleveland, OH).
Samples were drawn from the three reservoirs, air space instillate, pleural bath, and perfusate, at 10 and 70 min after starting the experiment protocol. To ensure homogeneous sampling from the air spaces, 2 ml of instillate were aspirated and reintroduced into the air spaces three times before removing each sample. This has been shown to provide a reproducibly mixed sample in our laboratory (25, 29, 34). All samples were centrifuged at 1,000 g for 15 min. Colorimetric analysis of the supernatant for EBD (absorbance at 620 nm) was performed in a Hitachi model U2000 spectrophotometer (Hitachi, San Jose, CA). Analysis of FITC-albumin (excitation 487 nm and emission 520 nm) was performed in a Perkin-Elmer fluorescence spectrometer (model LS-3B; Perkin-Elmer, Oakbrook, IL). 22Na+ and [3H]mannitol were measured in a beta-counter (Packard Tricarb, Downers Grove, IL). Na+ concentration was measured in an automated microprocessor-controlled analyzer employing the ion-selective electrode technique (Lytening 1; AMDEV, Danvers, MA).
Specific Protocols
Group A. We studied active Na+ transport and lung liquid clearance over 1 h in a control group of rat lungs (n = 10 animals).
Group B.
We established the dose-response curve of Iso
(104 M to
10
10 M) perfused through
the pulmonary circulation on lung liquid clearance (n = 6 animals in each group). The
doses of Iso selected for these studies were similar to the doses used
in other studies of ATII cells in our laboratory (27).
Group C. We determined the effects of Iso given in the instillate (n = 6 animals) and in both instillate and perfusate simultaneously (n = 6 animals) on active Na+ transport and lung liquid clearance.
Group D.
We studied the effects of a -adrenergic antagonist, propranolol
(10
5 M), on lung liquid
clearance in a control group (n = 4 animals) and lungs perfused with
10
6 M Iso
(n = 6 animals).
Group E.
To determine the possible role of
Na+ channels and Na-K-ATPase in
the stimulatory effects of Iso on liquid clearance, we studied the
effect of an Na-K-ATPase antagonist (5 × 104 M ouabain) administered
in the perfusate alone (n = 6 animals) or with 10
6 M Iso
(n = 6 animals), and we evaluated the
effect of an Na+-channel blocker
(10
4 M amiloride) added to
the alveolar instillate alone (n = 5 animals) or with 10
6 M Iso
(n = 6 animals).
Group F.
To examine the possible role of the intracellular microtubular
transport of ion-transporting proteins on the active
Na+ transport mediated by Iso, we
studied lung liquid clearance in rats treated with colchicine (0.25 mg/100 g body wt injected intraperitoneally ~15 h before the
experiments) without -agonist stimulation
(n = 6 animals) and with
10
6 M Iso added to the
perfusate (n = 5 animals). In
addition, eight animals were studied after intraperitoneal injection of
-lumicolchicine (0.25 mg/100 g body wt) ~15 h before
experiments with and without
-adrenergic stimulation. Lumicolchicine
is an isomer of colchicine that does not bind tubulin and does not
depolymerize microtubules (36). However, it shares other properties of
colchicine, such as inhibition of protein synthesis, and it is
therefore an appropriate control to demonstrate that the observed
effects of colchicine are due to microtubular disruption. The
inhibitory effect of colchicine but not lumicolchicine on microtubular
transport has been previously reported in bile secretion studies in
rats (8).
Group G.
To examine the possible association between the active
Na+ transport stimulated by Iso
and the intracellular microtubular transport, we studied the effect of
104 M amiloride added to
the alveolar instillate and 5 × 10
4 M ouabain added to the
perfusate in colchicine-treated rats without
-agonist stimulation
and with 10
6 M Iso added to
the perfusate (n = 6 animals in each
group).
Calculations
The alveolar lining fluid volume (VELF) was calculated by instilling 3 ml of fluid [initial volume (V0)] containing a known concentration of albumin tagged by EBD [(EBD)0] into the air space. After brief mixing, a sample was removed, and the EBD concentration at time t [(EBD)t] was determined. The mass of Evans blue-tagged albumin is the same in the instillate [V0(EBD)0] and in the lung after initial mixing [(V0 + VELF)(EBD)t]. Equating the two yields
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(1) |
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(2) |
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(3) |
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(4) |
As described (29), the passive movement of 22Na+, JNa,in, is given by
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(5) |
Similarly, the volume flux of mannitol [typically expressed as permeability of surface area (PSA)] is given by
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(6) |
Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appeared in the alveolar space during the experimental protocol. These calculations were carried out for each sampling period.
ATII Cell Isolation and Western Blot Analysis of Basolateral Membranes
ATII cells were isolated from normal and colchicine-treated rats as previously described (24, 28). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with 10 U/ml of elastase (Worthington Biochemical) for 20 min at 37°C. The tissue was minced and filtered through sterile gauze and 70-µm nylon mesh. The crude cell suspension was purified by differential adherence to IgG-pretreated dishes. ATII cells were homogenized in homogenization buffer, and basolateral membranes (BLMs) were obtained as described by Hammond et al. (14). After several centrifugations to discard the nuclear and mitochondrial pellet, the remaining supernatant was spun at 48,000 g for 30 min. Finally, the BLM fraction was recovered after the membrane pellet was centrifugated in a Percoll gradient (16%) at 48,000 g for 30 min. Na-K-ATPaseNa-K-ATPase Activity
Na-K-ATPase activity was determined in ATII cells isolated from control and colchicine-treated rats as the rate of [32P]ATP hydrolysis in suspended cells in buffer containing (in mM) 5 KCl, 10 MgCl2, 1 EGTA, 50 Tris · HCl, 3 Na2ATP (grade II, Sigma), and [32P]ATP (Amersham) in trace amounts. NaCl was added to a final concentration of 100 mM to examine activity at the concentration at which the Na-K-pump normally operates at maximal transport rate (>70 mM Na+). Cells were transiently permeabilized by thermic shock to make them permeable to [32P]ATP. The reaction was carried out for 15 min at 37°C with and without 1 µM Iso and terminated by placement on ice and addition of TCA-charcoal to absorb nonhydrolyzed [32P]ATP. The ouabain-insensitive ATPase activity was determined in buffer lacking Na+ and K+ but including 1 mM ouabain. Nonspecific ATP hydrolysis was determined in samples in the absence of cells. The 32Pi liberated was quantified by liquid scintillation counting (Packard Tricarb) and expressed as nanomoles of Pi per milligram of protein per minute.Data Analysis
Data are presented as mean values ± SE; n represents the number of animals in each experimental group. When comparisons were made between two experimental groups, an unpaired Student's t-test was used. When multiple comparisons were made, a one-way analysis of variance was used, followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results are considered significant at P < 0.05. ![]() |
RESULTS |
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Lung Liquid Clearance
The lungs of control rats instilled with 5 ml of buffered salt albumin solution cleared ~10% of the instillate in 1 h (0.45 ± 0.03 ml/h), whereas 10
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Propranolol (105 M)
administered in the instillate and perfusate simultaneously blocked the
effect of 10
6 M Iso on
active Na+ transport and lung
liquid clearance (0.59 ± 0.03 ml/h) but did not affect liquid
clearance in control lungs (0.47 ± 0.03 ml/h). Perfusion pressures
and flow rates in this group were not different from those in control
lungs.
Effects of Colchicine and Lumicolchicine
In colchicine-treated animals, active Na+ transport and lung liquid clearance significantly decreased compared with those in control lungs, and colchicine also partially inhibited the effect of Iso (Fig. 2). However, lung liquid clearance and Na+ transport were not decreased in rat lungs treated with
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Air space-instilled amiloride
(104 M) decreased the
active Na+ transport and lung
liquid clearance by ~46% compared with those of control lungs.
Amiloride (10
4 M) blocked
the stimulatory effect of Iso on lung liquid clearance without changing
the perfusion flow rates (Fig. 3).
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Ouabain (5 × 104 M)
perfused through the pulmonary circulation decreased lung liquid
clearance and Na+ transport by
~56% compared with control lungs and blocked the stimulatory effect
of Iso on lung liquid clearance without changing perfusion flow rates
(Fig. 4).
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In colchicine-treated rat lungs, amiloride
(104 M) added to the
instillate and ouabain (5 × 10
4 M) added to the
perfusate completely blocked basal and Iso-stimulated lung liquid
clearance (Figs. 3 and 4).
Effect of Iso on ATII Cells, Na-K-ATPase Activity, and
1-Subunit Protein Expression
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In parallel with these changes on lung liquid clearance and protein abundance in ATII cells treated with Iso, we observed a significant increase in the Na-K-ATPase activity in ATII cells isolated from control rats incubated for 15 min with 1 µM Iso (Fig. 6). Colchicine blocked the Na-K-ATPase activity stimulated by Iso.
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Epithelial Permeability
The movement of protein tracers across the alveolar epithelial barrier was similar to the rates reported in previous studies (3, 15, 29, 34). EBD-bound albumin instilled in the air space was not detected in the perfusate or bath compartments in any of the experimental groups. The movement of FITC-albumin from the pulmonary vascular compartment into the air spaces was not changed in any of the experimental groups. This minimal movement of albumin allows us to accurately assess lung liquid clearance in this model.As shown in Fig. 7, higher concentrations
of Iso (104 to
10
6 M) increased epithelial
22Na+
and [3H]mannitol
permeability. Propranolol, amiloride, ouabain, and colchicine did not
change the passive flux of small solutes (Table 1). The passive movement of
22Na+
and [3H]mannitol
across the membrane correlated closely with a slope of ~2.0 for
linear regression through the origin
(r = 0.77).
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DISCUSSION |
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The isolated perfused liquid-filled lung model is a relatively simple but powerful tool to investigate epithelial transport, but it has limitations that must be considered. The composition of alveolar instillate is arbitrary and may in some way alter the epithelial function. Also, it is not possible to determine which exact cell type is responsible for active transport and whether there is any contribution to transport by the airway epithelium. With consideration of the fact that the rate of active ion transport is low compared with passive bidirectional flux, very subtle variations may not be easily demonstrated (29). However, variables such as temperature, pH, hydrostatic and osmotic pressures, and electrolytes can be tightly controlled in this model (25, 29, 34). Although lungs are not normally filled with liquid, this model is appropriate for the study of lung edema clearance because it reproduces conditions observed during pulmonary edema in which a liquid-liquid interface occurs.
-Adrenergic agonists stimulate active
Na+ transport and edema clearance
in vivo (3), in isolated perfused lungs (7, 30), and in cultured
alveolar epithelial type II cells (6, 13). Iso is a nonselective
-adrenergic agonist with very low affinity for
-adrenergic receptors. In agreement with Saumon et al. (30), we have
confirmed that Iso increases active
Na+ transport and lung liquid
clearance in isolated perfused rat lungs by a similar magnitude as
shown for terbutaline and epinephrine (3, 7, 15). The effect of Iso on
lung liquid clearance was dose dependent (Fig. 1). The magnitude of
basal lung liquid clearance was similar to the rate reported
previously, ~10% over 1 h in control rat lungs (1, 10, 29, 30). We
determined that the
-adrenergic agonist Iso increased lung liquid
clearance via stimulation of
-adrenergic receptors as well as
amiloride-sensitive Na+ channels
and alveolar epithelial Na-K-ATPase. These data agree with previous
reports showing that
-adrenergic agonists regulate Na+ channels (37) and Na-K-ATPase
activity (27, 33) in ATII cells.
In a similar model, terbutaline exhibited greater effects on active
Na+ transport when it was placed
in the instillate (7), but in our study, the effects of Iso on active
Na+ transport were similar
regardless of whether it was added to the instillate, perfusate, or
both compartments. Therefore, -adrenergic agonists appear equally
efficient when placed in the air spaces or the pulmonary circulation,
suggesting either that receptors may be present on both sides of the
alveolar epithelium or that
-adrenergic agonists diffuse easily
across the alveolocapillary membrane.
In agreement with previous studies in vivo (3, 15) and in isolated
lungs (7), baseline lung liquid clearance and
Na+ transport were not affected by
the -antagonist propranolol. However, the experiments with the
-antagonist propranolol demonstrated that the effects of Iso on lung
liquid clearance were mediated via
-adrenergic receptors. Previous
studies have demonstrated that
-adrenergic agonists do not increase
Na+ transport in alveolar
epithelial type II cell monolayers (13).
Evidence for mechanisms by which Iso increased liquid clearance is
provided by the experiments with a nonselective
Na+-channel blocker (amiloride)
and an Na-K-ATPase antagonist (ouabain). Both drugs decreased the
Iso-stimulated Na+ transport and
lung liquid clearance, suggesting that the stimulatory effect of Iso on
edema clearance is linked to Na+
transport via the amiloride-sensitive pathway
(Na+ channels) and Na-K-ATPase. In
agreement with our findings, -adrenergic agonists
stimulated the dome formation and short-circuit currents across
alveolar epithelial type II cells (13).
The inhibition by amiloride of lung liquid clearance in our study was
similar to previous reports (1, 10, 15, 30). The effect of amiloride at
these concentrations (104
M) probably includes the inhibitory effects on apical
Na+ channels, on the
Na+-H+
antiport system, and possibly on the basolaterally located Na-K-ATPase (2, 32). However, it does not affect the
Na+-glucose cotransport. It has
been previously reported that when glucose is replaced by mannitol in
the instillate, amiloride almost completely inhibits fluid absorption
in isolated perfused lung (30).
Na-K-ATPase and ion channels are associated in epithelial cells with
proteins (e.g., actin, ankyrin, and fodrin) that constitute the
cortical cytoskeleton, which provides the foundation for spatial distribution of proteins within the plasma membrane (22). Evidence is
now emerging for a dynamic regulation of
Na+ channels and Na-K-ATPase
activity by constituents of the cytoskeleton. In addition to governing
cell structure and motility, actin appears to also serve as a signaling
molecule required in Na+-channel
activation by cAMP-dependent protein kinase (26). Also, there is
evidence that Na-K-ATPase exists in intracellular pools and, on
specific signaling (i.e., -adrenergics), can be rapidly recruited
via cell microtubular transport into the plasma membrane (5).
Therefore, we studied whether inhibition of microtubular transport to
the plasma membrane by colchicine would inhibit the stimulatory effects
of Iso on active Na+ transport and
edema clearance. Colchicine inhibited lung liquid clearance and
Na+ transport in control lungs and
rat lungs stimulated by Iso, whereas the isomer
-lumicolchicine,
which shares many colchicine properties with the exception of
inhibiting microtubular transport (36), did not inhibit the Iso
stimulation of lung liquid clearance. Colchicine decreased basal lung
clearance, confirming that the cortical cytoskeleton and intracellular
microtubular transport are structurally and functionally linked to
Na+ channels and Na-K-ATPase
function in polarized epithelial cells (4, 5, 26, 31). The Na-K-ATPase
- and
-subunits are degraded with a half-life of 10-12 h
(16), and in our study, colchicine was administered ~15 h before the
experiments; therefore, basal lung liquid clearance probably decreased
because the microtubular transport pathway was disrupted, inhibiting
the movement of Na+-transporting
proteins to the plasma membrane (Figs. 2 and 5).
We observed that an Na+-channel blocker (amiloride) and an Na-K-ATPase antagonist (ouabain) completely blocked lung edema clearance stimulated by Iso in colchicine-treated rats (Figs. 2-4). Thus the inhibitory effect of colchicine on the Iso stimulation of lung liquid clearance is probably due to disruption of the vectorial Na+ transport pathway. In agreement with our results, others have reported the modulatory role of the cell microtubular transport and the cytoskeleton on the apical Na+-channel and Na-K-ATPase activity in basal and stimulated conditions (4, 5, 26, 31).
-Adrenergic agonists that stimulate Na-K-ATPase activity by
increasing cellular cAMP might achieve this effect by interacting with
the cell microtubular transport pathway (4, 5, 27). To examine this
association, we studied the Na-K-ATPase activity and
1-subunit protein abundance in
ATII cells isolated from control and colchicine-treated rat lungs. We
observed that Iso increased Na-K-ATPase activity and
1-subunit abundance in BLMs of
alveolar epithelial type II cells and that this effect was completely
abolished by colchicine (Figs. 5 and 6). We thus reason that cell
microtubular transport of Na+
pumps to the cell plasma membrane has a role in active
Na+ transport in basal conditions
and during stimulation of lung edema clearance by
-adrenergic
agonists.
Na-K-ATPase synthesis occurs in polysomes that become related to the
membrane of the rough endoplasmic reticulum, and the -subunits
are transported through a network of membrane compartments to their
functional site in the plasma membrane (5, 23). The Na-K-ATPase
upregulation (activity measured by ATP hydrolysis and protein
expression in BLMs by Western blot) by Iso occurs in ~15 min in ATII
cells (27), and we have demonstrated that Iso increased active
Na+ transport in isolated rat
lungs within 1 h (Fig. 1). The increase in
Na+ transport in our model is due
to upregulation of apical Na+
channels and mobilization of Na+
pumps from intracellular pools by translocation to the plasma membrane
via cell microtubular transport and possibly other posttranslational events.
In agreement with a previous study (30), we found that the paracellular permeability of the small molecules (22Na+ and [3H]mannitol) was increased by high concentrations of Iso. Changes in the paracellular pathway in epithelia secondary to the action of either catecholamines (21) or cAMP (9) have been reported previously. However, even at higher doses, Iso did not significantly change epithelial permeability to albumin; thus it did not interfere with our ability to estimate the rates of lung liquid clearance.
Restoration of alveolar epithelial permeability and lung edema clearance in patients with acute hypoxemic respiratory failure has been associated with improved clinical outcome (20, 35). In two different models of hyperoxic lung injury, changes in active Na+ transport and lung edema clearance paralleled changes in Na-K-ATPase function in ATII cells (24, 25, 34). Therefore, it is possible that Iso could be utilized as a therapeutic tool to upregulate Na-K-ATPase function and active Na+ transport in patients with pulmonary edema and hypoxemic respiratory failure.
In summary, our data show that Iso increases active
Na+ transport and lung liquid
clearance in a dose-dependent manner. This stimulation is mediated by
the -adrenergic pathway in which intracellular microtubular
transport of Na+ pumps from inner
pools to the cell plasma membrane appears to play an important role.
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ACKNOWLEDGEMENTS |
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This research was supported, in part, by National Heart, Lung, and Blood Institute Grant HL-48129, American Heart Association Grant 96012890, the Department of Medicine of Michael Reese Hospital, and Pontificia Universidad Católica de Chile.
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FOOTNOTES |
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Address for reprint requests: J. I. Sznajder, Div. of Pulmonary and Critical Care Medicine, Michael Reese Hospital and Medical Center, 2929 S. Ellis Ave., RC-216, Chicago, IL 60616.
Received 19 May 1997; accepted in final form 4 February 1998.
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