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1 Department of Animal Physiology, Lund University, S-223 62 Lund, Sweden; 3 Cardiovascular Research Institute, University of California San Francisco, San Francisco, California 941434-0130; 2 Division of Gastroenterology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205-2195; and 4 Department of Physiology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio 44272-0095
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The contributions of
amiloride-sensitive and -insensitive fractions of alveolar fluid
clearance in adult ventilated rats were studied under control
conditions and after 
-adrenergic stimulation. Rats were
instilled with a 5% albumin solution containing terbutaline (10
4 M) or dibutyryl-cGMP (DBcGMP; 104
M) with or without the cyclic nucleotide-gated cation channel inhibitor
l-cis-diltiazem (103 M) and/or
amiloride (103 M). Alveolar fluid clearance over 1 h
was 18 ± 2% in controls. In controls, amiloride inhibited
46 ± 15% of alveolar fluid clearance, whereas
l-cis-diltiazem had no inhibitory effect.
Terbutaline and DBcGMP stimulated alveolar fluid clearance by 85 ± 3 and 36 ± 5%, respectively. Amiloride and
l-cis-diltiazem inhibited nearly equal fractions
of terbutaline-stimulated alveolar fluid clearance when given alone.
Amiloride and l-cis-diltiazem given together inhibited a significantly larger fraction of alveolar fluid clearance in terbutaline-stimulated rats and in DBcGMP-stimulated rats. Based on
these data, terbutaline stimulation recruited both amiloride-sensitive and l-cis-diltiazem-sensitive pathways. In
contrast, DBcGMP mainly recruited
l-cis-diltiazem-sensitive pathways. Therefore,
the amiloride-insensitive fraction of Na+-driven alveolar
fluid clearance may be partly mediated through cyclic nucleotide-gated
cation channels and activated by an increase in intracellular cGMP.
l-cis-diltiazem; cyclic nucleotide-gated cation channels; dibutyryl-guanosine-3'5'-cyclic monophosphate; epithelial sodium ion channels; terbutaline
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ALVEOLAR FLUID CLEARANCE
IS driven by vectorial Na+ transport across the
alveolar epithelium (5, 10, 13, 16, 17, 19, 27, 30, 38,
39). Na+ enters the alveolar epithelial cells
through apically located Na+ channels and is
subsequently extruded by basolaterally located Na+-K+-ATPases (for reviews, see Refs.
24, 26). Increased levels of intracellular
cAMP stimulate alveolar fluid clearance (3). Substances
that increase intracellular cAMP include epinephrine (10, 16,
30) and various other -adrenergic agonists (5, 19, 30,
31, 38). The mechanism by which intracellular cAMP stimulates
alveolar fluid clearance is not fully understood, but cAMP could act in
multiple ways, e.g., by recruiting
Na+-K+-ATPases to cell membranes
(31), by recruiting Na+ channels [epithelial
Na+ channels (ENaC)] to the apical membrane
(35), and/or by increasing the open probability of the
Na+ channel (2, 3, 24, 26). Also, recent data
suggest that chloride may play a role in -agonist-mediated
upregulation of transport across alveolar epithelial type II cells
(20). Substances that do not interact with -adrenergic
receptors also regulate alveolar fluid clearance, e.g., corticosteroids
(17, 29) and growth factors (19, 36, 38).
In several animal species, the Na+ channel blocker amiloride inhibited a significant fraction of unstimulated and stimulated alveolar fluid clearance (5, 10, 13, 16, 17, 19, 30, 38, 39). The amiloride sensitivity may be related to the ENaC (2, 16, 29, 38, 39) and possibly also to nonspecific cation channels (23, 37). In contrast, the amiloride-insensitive fraction of alveolar fluid clearance and Na+ absorption is less well understood. Some recent studies demonstrated that the amiloride-insensitive fraction of 8-bromo-cGMP-stimulated short-circuit current and 22Na+ uptake in rat tracheal epithelia was inhibited by dichlorobenzamil or l-cis-diltiazem, which are both inhibitors of cyclic nucleotide-gated cation (CNG) channels (33). In subsequent studies, dichlorobenzamil inhibited a significant fraction of lung fluid absorption in sheep (21), which suggested that CNG channels play a role in alveolar fluid absorption. The CNG channels were originally identified and cloned from vertebrate rod photoreceptors (15) and the olfactory neuroepithelium (28). Three different CNG channel isoforms have been cloned (6). The CNG1 channel has a wide tissue distribution and is localized to eye, brain, thymus, heart, and lung (12). In the lung, mRNA for CNG1 has been localized to the distal lung epithelium and mainly to alveolar epithelial cells.
We hypothesized that the amiloride-insensitive fraction of alveolar
fluid clearance in the rat is, at least partly, mediated through the
CNG channels. Therefore, our first aim was to investigate the
functional contributions of amiloride-sensitive Na+
channels and amiloride-insensitive CNG channels for alveolar fluid
clearance under unstimulated conditions. Because CNG channels are
stimulated by cyclic nucleotides and -adrenergic agonists act
through the increase of both intracellular cAMP and cGMP
(32), our second aim was to investigate
amiloride-sensitive and amiloride-insensitive fractions of alveolar
fluid clearance after -adrenergic stimulation. Our third aim was to
investigate whether direct intrapulmonary instillation of
dibutyryl-cGMP (DBcGMP) stimulated alveolar fluid clearance and to
study amiloride-sensitive and amiloride-insensitive fractions of
alveolar fluid clearance after DBcGMP instillation.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Adult male Sprague-Dawley rats (n = 69), weighing 275-350 g (B&K Universal, Sollentuna, Sweden), were used. The rats were kept at a 12:12-h day-night rhythm and had free access to standard rat chow (R3; Astra-Ewos, Södertälje, Sweden) and tap water. The Ethical Review Committee on Animal Experiments at Lund University approved the experiments.Preparation of Solutions
A 5% albumin instillate solution was prepared by dissolving 50 mg/ml bovine serum albumin (Sigma Chemical, St. Louis, MO) in 0.9% NaCl (Pharmacia-UpJohn, Uppsala, Sweden). In some studies, 104 M terbutaline hemisulfate (terbutaline; Sigma
Chemical) or 104 M DBcGMP (Sigma Chemical) plus
104 M aminophylline (Sigma Chemical) were added to the
albumin solution. Also, 103 M amiloride hydrochloride
hydrate (amiloride; Sigma Chemical) and/or 103 M
l-cis-diltiazem hydrochloride
(l-cis-diltiazem; Research Biochemicals, Natick,
MA) were added to the instillate solution in some studies. For the
DBcGMP studies, an aminophylline infusion solution (104 M
in 0.9% NaCl) was prepared.
Surgical Procedure and Ventilation
The rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt; Apoteksbolaget, Umeå, Sweden). A 2.0-mm (ID) endotracheal tube (PE-240, Clay Adams, Becton Dickinson, Sparks, MD) was inserted through a tracheotomy, and a 0.58-mm (ID) catheter (PE-50, Clay Adams, Becton Dickinson) was inserted in the left carotid artery. Another PE-50 catheter was inserted in the left jugular vein for administration of aminophylline during the DBcGMP studies. Pancuronium bromide (0.3 mg · kg body wt1 · h1; Pavulon, Organon Teknika,
Boxtel, The Netherlands) was administered through the arterial catheter
for neuromuscular blockade. Pupil dilatation, blood pressure, and heart
rate were used as indicators of anesthesia. The anesthesia was
complemented when necessary, e.g., if blood pressure began to increase
significantly. The rats were maintained in the left lateral decubitus
position during the experiment and were ventilated with a
constant-volume piston pump (Harvard Apparatus, Nantucket, MA) with an
inspired oxygen fraction of 1.0 and tidal volumes set to reach peak
airway pressures of 10-12 cmH2O during the baseline
period. Positive end-expiratory pressure was kept at 2-3
cmH2O.
Peak airway pressure, arterial blood pressure, and heart rate were measured with calibrated pressure transducers (UFI model 1050BP or TSD104, BioPac Systems, Goleta, CA) connected to analog-to-digital converters and amplifiers (MP100 and DA100, respectively, BioPac Systems) and continuously recorded with Acknowledge 3.2 software (BioPac Systems) on an IBM PC-compatible computer.
General Protocol
After a 30-min baseline period of stable heart rate and blood pressure, the instillation tubing (PE-50) was passed through the tracheal tube into the left lung without interrupting ventilation. The instillation solution (3-4 ml/kg body wt) was instilled over 25 min by infusing 0.04 ml/min with a 1-ml syringe. The study proceeded over 1 h and started at the beginning of fluid instillation. After instillation, the tubing was withdrawn. For the DBcGMP studies, a bolus dose of 0.56 mg/kg body wt aminophylline in 0.4 ml was injected through the vein catheter 5 min before instillation. Every 12 min until 48 min after the start of the experiment, the initial aminophylline dose was complemented with 21 mg/kg body wt aminophylline in 0.15 ml of the infusion solution. This protocol gave a total dose of 140 mg · kg body wt1 · h1. At
the end of the experiment, a blood sample (2 ml) was withdrawn from the
carotid artery. The abdomen was opened, and the rats were exsanguinated
by transection of the renal artery. The lungs were removed from the
chest through a midline sternotomy. A PE-50 catheter was passed into
the instilled lung, and a sample of the remaining alveolar fluid was
collected. A previous study demonstrated that the protein concentration
in fluid aspirated with a catheter wedged into the distal air spaces is
a good reflection of the alveolar fluid protein concentration
(4). Protein concentrations in the instilled solutions and
in the final alveolar fluid samples were measured
spectrophotometrically (iEMS reader MF, Labsystems, Helsinki, Finland)
with the Lowry method (22) adapted for microtiter plates.
Specific Protocol
All rats were surgically prepared as described above. The rats were randomly divided into the following groups. All rats were studied for 1 h as described in General Protocol.Control studies. The rats (n = 6) were instilled with the 5% albumin.
Terbutaline studies.
The rats (n = 6) were instilled with the 5% albumin
solution containing 104 M of the -adrenergic agonist terbutaline.
DBcGMP studies.
The rats (n = 5) were instilled with the 5% albumin
solution containing 104 M of the membrane-permeable cGMP
analog DBcGMP plus 104 M aminophylline. Aminophylline was
added to the instillate to prevent hydrolysis of DBcGMP. When DBcGMP
was instilled, aminophylline was given intravenously during the 1-h
study. To test that aminophylline did not affect alveolar fluid
clearance by itself, another group (n = 3) with
104 M aminophylline added to the albumin solution was studied.
l-cis-Diltiazem studies.
The rats were instilled with the 5% albumin solution containing
103 M of the CNG channel inhibitor
l-cis-diltiazem (n = 6) or
103 M l-cis-diltiazem plus
104 M terbutaline (n = 5). Another group
of rats was instilled with 5% albumin solution containing
103 M l-cis-diltiazem plus
104 M DBcGMP plus 104 M aminophylline
(n = 5).
Amiloride studies.
The rats were instilled with the 5% albumin solution containing
103 M of the sodium channel inhibitor amiloride
(n = 6) or 103 M amiloride plus
104 M terbutaline (n = 4). Another group
of rats was instilled with 5% albumin solution containing
103 M amiloride plus 104 M DBcGMP plus
104 M aminophylline (n = 5).
l-cis-Diltiazem plus amiloride studies.
The rats were instilled with the 5% albumin solution containing
103 M l-cis-diltiazem plus
103 M amiloride (n = 6) or containing
103 M l-cis-diltiazem plus
103 M amiloride plus 104 M terbutaline
(n = 7). Another group of rats was instilled with 5%
albumin solution containing 103 M
l-cis-diltiazem plus 103 M
amiloride plus 104 M DBcGMP plus 104 M
aminophylline (n = 5).
Alveolar Fluid Clearance Analysis
Alveolar fluid clearance was calculated from the increase in alveolar albumin concentration over the 1-h study, as has been done in several studies before (10, 16, 17, 29, 30). Data are presented in two ways. First, alveolar fluid clearance is presented as the final-to-instilled protein concentration ratio, i.e., the ratio of the final alveolar fluid sample protein concentration over the instilled protein concentration. This provides direct evidence for clearance of excess fluid from the distal air spaces. Because there were no changes in epithelial protein permeability in any experimental group and little protein left the air spaces, the increase in protein concentration over 1 h is caused by water leaving the air spaces (data not shown). The second way of presenting the data is by calculating alveolar fluid clearance (AFC) by the following equation
![AFC<IT>=</IT>[(V<SUB>I</SUB><IT>−</IT>V<SUB>F</SUB>)<IT>/</IT>V<SUB>I</SUB>]<IT>×100</IT>](/content/vol90/issue4/fulltext/1489/img001.gif)
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(1) |
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(2) |
Calculation of Fractional Inhibition of Alveolar Fluid Clearance
The fractional inhibition of alveolar fluid clearance by amiloride and l-cis-diltiazem under control or stimulated conditions was calculated by the following equation
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(3) |
Alveolar Fluid Clearance Composition
Alveolar fluid clearance was also analyzed with respect to absolute alveolar fluid clearance (%), which constituted the alveolar fluid clearance that was sensitive to amiloride or l-cis-diltiazem. The amiloride-sensitive alveolar fluid clearance was calculated as the difference between control (or stimulated) alveolar fluid clearance and the alveolar fluid clearance after amiloride inhibition. To compensate for overlapping inhibition by the two drugs, we calculated the l-cis-diltiazem-sensitive fraction as alveolar fluid clearance after amiloride plus l-cis-diltiazem inhibition subtracted from alveolar fluid clearance after amiloride inhibition.Statistics
Data are summarized and presented as means ± SD. The data were analyzed by one-way ANOVA with Tukey's test post hoc. Differences were considered significant when a P value of <0.05 was obtained.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Influence of CNG Channel Inhibitors Under Unstimulated Conditions
We investigated the effect of the CNG channel inhibitor l-cis-diltiazem on the unstimulated alveolar fluid clearance (control) in adult ventilated rats. When 103 M l-cis-diltiazem were added to
the 5% albumin solution, no inhibition of control alveolar fluid
clearance was observed (Fig. 1, Table 1). On the other hand, amiloride
(103 M) significantly inhibited alveolar fluid clearance
by 46 ± 15%. The combination of amiloride and
l-cis-diltiazem had no additive effect (Table 1).
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Influence of CNG Channel Inhibition Under Terbutaline Stimulation
We then investigated whether terbutaline stimulation (104 M) of alveolar fluid clearance altered the
composition of amiloride-sensitive and amiloride-insensitive pathways
of fluid clearance from the distal air spaces. Terbutaline stimulated
alveolar fluid clearance by 85 ± 6% over control conditions, a
result similar to previous studies in the rat (17, 38).
Amiloride inhibited alveolar fluid clearance under stimulated
conditions and returned the clearance to control levels (Fig.
2, Table 1). When
l-cis-diltiazem was added to the instillate, the
alveolar fluid clearance again similarly decreased to control levels,
i.e., the terbutaline stimulation was completely abolished (Fig. 2,
Table 1). Amiloride and l-cis-diltiazem administered together further decreased alveolar fluid clearance to
below control levels (Fig. 2, Table 1).
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Influence of CNG Channel Inhibition Under DBcGMP Stimulation
It was not previously known if alveolar fluid clearance could be stimulated by cGMP. Therefore, we studied alveolar fluid clearance after instillation of a membrane-permeable cGMP analog. Addition of DBcGMP to the instilled fluid significantly increased alveolar fluid clearance by 36 ± 5% compared with control rats (Fig. 3, Table 1). However, DBcGMP at this concentration (104 M) did not increase alveolar fluid
clearance as effectively as terbutaline did (Table 1). Because the CNG
channels are activated by cGMP (6), we investigated
whether inhibition of alveolar fluid clearance by
l-cis-diltiazem was altered after simultaneous administration of DBcGMP. Both l-cis-diltiazem
and amiloride inhibited the cGMP-stimulated alveolar fluid clearance to
control levels (Fig. 3). The combination of
l-cis-diltiazem and amiloride was additive,
because the combination of both inhibitors decreased the
cGMP-stimulated alveolar fluid clearance to levels significantly below
control alveolar fluid clearance (Fig. 3).
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Fractional Inhibition of Alveolar Fluid Clearance
The fractional inhibition of alveolar fluid clearance by the channel blockers amiloride and l-cis-diltiazem was calculated for both the control and stimulated conditions (Table 1; Fig. 1). Amiloride was the most effective inhibitor of alveolar fluid clearance under control conditions, whereas l-cis-diltiazem had no inhibitory effect on control alveolar fluid clearance.When alveolar fluid clearance was stimulated by terbutaline, both blockers significantly inhibited alveolar fluid clearance. The most dramatic change was with l-cis-diltiazem, which, during control conditions, had no inhibitory effect on alveolar fluid clearance but, during terbutaline-stimulated conditions, inhibited ~50% of the alveolar fluid clearance. The combination of amiloride and l-cis-diltiazem increased the fractional inhibition to ~80%, which was greater than that of either of the inhibitors given alone (Table 1).
On stimulation of alveolar fluid clearance with DBcGMP, l-cis-diltiazem inhibited ~25% of alveolar fluid clearance (Table 1). On the other hand, amiloride inhibited only ~30% of alveolar fluid clearance, which was less than control or terbutaline-stimulated alveolar fluid clearance. l-cis-Diltiazem and amiloride in combination were additive and inhibited a larger fraction (~50%) of alveolar fluid clearance compared with the administration of each drug alone.
Ratio of Amiloride-sensitive and -insensitive Alveolar Fluid Clearance
The amiloride-sensitive and l-cis-diltiazem-sensitive (amiloride-insensitive) components of alveolar fluid clearance are illustrated in Fig. 4. After stimulation with terbutaline, the amiloride-sensitive component increased twofold compared with control rats, whereas the amiloride-insensitive component increased fivefold. After DBcGMP stimulation, the amiloride-sensitive component remained unchanged, whereas the amiloride-insensitive component increased threefold compared with control rats. The component that was insensitive to either of the drugs remained constant, irrespective of treatment.
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Airway Pressure and Hemodynamics
Airway pressure did not vary within groups or between groups during the experiments. Blood pressure increased <22% from baseline during the initial phase of instillation in all groups but returned to baseline values within 5-10 min. Furthermore, blood pressure varied <18% among groups at any given time point. Heart rate never varied by >12% within groups over the experimental time period and by <13% among groups at any given time point. At the end of the experiment, the hematocrit was always within the normal range and varied <11% within groups and <13% among groups.| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Based on several in vivo studies in several different animal species, amiloride inhibits 30-90% of alveolar fluid clearance (1, 5, 10, 13, 16, 17, 19, 21, 29, 30, 38, 39). In the rat, 45% is amiloride sensitive; the amiloride-insensitive fraction of alveolar fluid clearance has not been fully investigated. In this study, we hypothesized that part of the amiloride-insensitive fraction of alveolar fluid clearance in adult rats was mediated through the CNG channel that has been localized to the adult rat lung (12). The CNG channels have also recently been shown to play a role in the clearance of alveolar fluid in sheep (21). Therefore, we used the CNG channel blocker l-cis-diltiazem to evaluate the contribution of these channels to alveolar fluid clearance under normal and stimulated conditions in rats.
We used relatively high doses of amiloride and
l-cis-diltiazem (103 M). We
selected these concentrations because both
l-cis-diltiazem and amiloride are
low-molecular-weight compounds and amiloride has previously been used
at this concentration in several studies of in vivo alveolar fluid
clearance (1, 5, 10, 16, 17, 29, 30, 38, 39). Also, it has
been demonstrated that a fraction of amiloride binds to albumin and
another fraction leaves the air spaces rapidly because of the small
molecular size (39). Therefore, although a relatively high
concentration is added, the effective in vivo concentrations were
probably much lower.
Amiloride inhibited 40-50% of alveolar fluid clearance under normal and stimulated conditions, an inhibition well in accordance with what has been reported previously in the rat (10, 17, 19, 38, 39). l-cis-Diltiazem lacked an inhibitory effect when it was added to the instilled fluid under control conditions, whereas this drug decreased the terbutaline-stimulated alveolar fluid clearance to a level similar to that of amiloride. When l-cis-diltiazem was administered together with amiloride, an almost complete blockade of alveolar fluid clearance was observed. This result suggests that terbutaline stimulation recruits or activates pathways for alveolar fluid clearance in addition to, and perhaps different from, the ENaC, possibly CNG channels. These inhibitor studies cannot exclude the possibility that l-cis-diltiazem may act on the same pathway as amiloride does. However, because l-cis-diltiazem did not inhibit control alveolar fluid clearance, this result supports the hypothesis that terbutaline stimulation activates or recruits an amiloride-insensitive Na+ transporting pathway of alveolar fluid clearance. Alternatively, terbutaline may have altered the constitution of ENaC, making the ENaC channel sensitive to l-cis-diltiazem. In fact, there are reports suggesting that ENaC structure may be changed under certain conditions, and thus amiloride sensitivity could be altered (9). Also, in a mutation of ENaC causing pseudohypoaldosteronism, the amiloride sensitivity was significantly reduced (7). However, because l-cis-diltiazem is an inhibitor of the CNG channel, it is more likely that terbutaline recruited or activated CNG channels in the distal lung epithelium together with amiloride-sensitive pathways.
When alveolar fluid clearance was stimulated with terbutaline, the fractional inhibition (presented as percentage of stimulated alveolar fluid clearance) by amiloride was not significantly altered compared with control conditions (Table 1). In contrast, the fractional inhibition by amiloride after DBcGMP stimulation decreased compared with control conditions. These results indicate that the composition of amiloride-sensitive and l-cis-diltiazem-sensitive pathways for alveolar fluid clearance was altered differently after stimulation by terbutaline and DBcGMP, respectively. Because of this, we presented the effect of the inhibitors as a component of each pathway. We calculated the composition of alveolar fluid clearance that represents each pathway (amiloride sensitive, l-cis-diltiazem sensitive, and noninhibited pathways) (Fig. 4). With this approach, there were differences among control, terbutaline-stimulated, and DBcGMP-stimulated conditions. As shown in Fig. 4, the amiloride-sensitive component increased twofold after terbutaline stimulation compared with control, but the fractional percentage of inhibition by amiloride remained unchanged because the alveolar fluid clearance also doubled (Table 1). Corresponding calculations for l-cis-diltiazem inhibition after terbutaline stimulation, compared with control conditions, gave a fivefold increase in inhibition. These data suggest that terbutaline might have recruited similar absolute components of amiloride-sensitive and l-cis-diltiazem-sensitive pathways for alveolar fluid clearance in the rat. However, after DBcGMP stimulation of alveolar fluid clearance, the l-cis-diltiazem-sensitive alveolar fluid clearance increased by threefold, which was less than the increase after terbutaline stimulation. By contrast, the amiloride-sensitive alveolar fluid clearance remained unchanged after DBcGMP stimulation.
On balance, the results suggest that both amiloride-sensitive sodium channels and CNG channels were activated after terbutaline stimulation of alveolar fluid clearance in the rat, because amiloride and l-cis-diltiazem both inhibited alveolar fluid clearance. By contrast, after DBcGMP stimulation of alveolar fluid clearance, only the l-cis-diltiazem-sensitive component of alveolar fluid clearance increased. In view of these results and because terbutaline may increase both intracellular cGMP and cAMP (32), the results suggest that terbutaline may have stimulated alveolar fluid clearance in two different ways. First, cGMP may stimulate alveolar fluid clearance by activation of CNG channels. Second, an amiloride-sensitive fraction of alveolar fluid clearance (probably mostly ENaC) is mediated through intracellular cAMP (3, 16, 19, 30).
Because other stereoisomers of diltiazem are inhibitors of the voltage-gated Ca2+ channel and because CNG channels can cause Ca2+ influx (32), it is also possible that l-cis-diltiazem affected intracellular Ca2+ levels. Terbutaline activates amiloride-sensitive Na+ uptake in isolated fetal distal lung epithelial cells (37) and adult epithelial alveolar type II cells (14) in a way that may involve increased intracellular Ca2+ concentration. Such an increase could be mediated through uptake of extracellular Ca2+ via Ca2+ channels and/or recruitment from intracellular stores. If l-cis-diltiazem inhibits Ca2+ channels, the inhibition of alveolar fluid clearance by l-cis-diltiazem after terbutaline stimulation could occur because terbutaline fails to stimulate ENaC recruitment and/or activation because of the inability of terbutaline to increase intracellular Ca2+.
A certain fraction of alveolar fluid clearance is not inhibited by
either amiloride or l-cis-diltiazem under control
conditions or after stimulation. This fraction of alveolar fluid
clearance may be explained in at least three possible ways. First, a
third yet unidentified Na+-specific channel or a
nonspecific cation channel could mediate this fraction of alveolar
fluid clearance. Several reports have suggested the existence of
different cation channels in the alveolar epithelium (for review, see
Ref. 24). However, most of these channels are inhibited by
amiloride at the concentrations used in this study and would,
therefore, probably be included in the fraction of alveolar fluid
clearance that is sensitive to amiloride. Second, transport of
Cl across the alveolar epithelium could also contribute
to the driving of water from the air spaces. It has been suggested that
the cystic fibrosis transmembrane conductance regulator channel may
regulate absorption of Na+ in the adult lung
(20). There is also preliminary evidence that the
bumetanide-sensitive 2Cl-Na+-K+
cotransporter might be involved in stimulated alveolar fluid clearance
(18). Third, passive forces, i.e., nonmetabolic pathways, could constitute part of the unstimulated alveolar fluid clearance mechanism. However, this pathway is probably very small, especially in
the in vivo lung, compared with the active pathway (27). Therefore, it is likely that the remaining alveolar fluid clearance after amiloride or l-cis-diltiazem inhibition is
mediated through still unknown pathways.
In this study, 48 ± 3% of alveolar fluid clearance could be
inhibited by amiloride and 48 ± 21% by
l-cis-diltiazem after -adrenergic stimulation.
In various in vitro preparations (single cells or monolayers of
alveolar epithelial type II cells or fetal distal lung epithelial
cells), up to 100% of Na+ channel activity can be
inhibited with amiloride (for review, see Ref. 24). The
discrepancy between the in vivo and in vitro situation might be
explained by heterogeneous distribution of different Na+ or
cation channels in the alveolar epithelium as well as by cellular changes after isolation and culture conditions. Previously, it was
demonstrated that the CNG1 channel was located mainly in the alveolar
epithelial cells of the lung (12). 
-ENaC mRNA and ENaC
protein have been localized primarily to the alveolar epithelial type
II cells in the deep lung and to airway epithelial cells in adult rats
(25). Consequently, as suggested previously
(33), if the
l-cis-diltiazem-sensitive CNG1 channels were
located primarily on alveolar epithelial type I cells and the
amiloride-sensitive ENaC were located primarily on the alveolar type II
cells, then this could explain why amiloride does not inhibit 100% of
alveolar fluid clearance in vivo in most species. In contrast to the
alveolar type II cell preparations, the in vivo condition also includes alveolar type I cells. Hence, significant portions of
amiloride-insensitive Na+ (or cation) channels in the
alveolar type I cells would not be included in those alveolar type II
cell preparations and, therefore, could explain why amiloride fails to
inhibit >40% of alveolar fluid clearance in the in vivo lung. Thus
terbutaline may increase ENaC activity or the quantity of active ENaC
in the membrane of the alveolar type II cells simultaneously with
activation of CNG1. It has, however, been suggested that ENaC could be
present in alveolar epithelial type I cells also (8). Our
hypothesis that a primary localization of CNG1 channels to the alveolar
type I cells and that these channels mediate a fraction of stimulated alveolar fluid clearance is also supported by the fact that guanylate cyclase exists in alveolar type I cells (34), where the
CNG1 mRNA is abundantly expressed (12).
Vectorial transport of ions requires an entry step at the apical cell
membrane and an exit step at the basolateral cell membrane. In the
alveolar epithelium, the entry step is ENaC and the exit step is the
basolateral Na+-K+-ATPase. In this study, we
focused our work on apical amiloride-sensitive and -insensitive
Na+ channels and their role in driving alveolar fluid
clearance in adult rat lungs. It is likely that both the apical entry
step (Na+ channels) and the basolateral exit step
(Na+-K+-ATPase) are linked to be able to
accommodate changes in fluid transport across the alveolar epithelium.
Although not studied here, effects of
l-cis-diltiazem or amiloride on the lung
epithelial Na+-K+-ATPase function cannot be
ruled out. There is some evidence that the
Na+-K+-ATPase function is upregulated by
-adrenergic agonists and cGMP-dependent protein kinases (11,
31). Although it is more likely that l-cis-diltiazem inhibits CNG channels, limited
inhibitory effects on the basolateral
Na+-K+-ATPase cannot be ruled out.
We conclude that, under normal conditions in in vivo rat lungs, Na+-driven alveolar fluid clearance is mediated through amiloride-sensitive pathways, probably ENaC, and a yet incompletely characterized amiloride-insensitive pathway. Stimulation with terbutaline may recruit or activate both ENaC and CNG channels in the alveolar epithelium, resulting in an increased alveolar fluid clearance. Terbutaline could act by increasing intracellular cAMP, stimulating ENaC channels (3, 16, 19, 30), and also increasing intracellular cGMP, thus stimulating CNG channels in the alveolar epithelium.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants HL-51854 and DK-48977, Swedish Natural Science Council, Crafoord Foundation for Scientific Research, Magnus Bergwall's Foundation, and the Royal Physiographic Society, Lund, Sweden.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. G. Folkesson, Associate Professor, Dept. of Physiology, Northeastern Ohio Universities College of Medicine (NEOUCOM), 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272-0095 (E-mail: hgfolkes{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.
Received 7 June 2000; accepted in final form 3 November 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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terbutaline, and
terbutaline + l-cis-diltiazem (1-way
ANOVA with Tukey's post hoc test).
