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Am J Physiol Lung Cell Mol Physiol 291: L610-L618, 2006. First published May 5, 2006; doi:10.1152/ajplung.00426.2005
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Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations

My N. Helms,1,3 Julie Self,1,3 Hui Fang Bao,1,3 Lauren C. Job,1,3 Lucky Jain,1,2,3 and Douglas C. Eaton1,2,3

Departments of 1Physiology and 2Pediatrics and 3The Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia

Submitted 5 October 2005 ; accepted in final form 6 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-µm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2 ± 2.5 and 22 ± 3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 µM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31 ± 0.19 to 0.60 ± 0.21 (P < 0.001). The D1 receptor antagonist SCH-23390 (10 µM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.

AT1 cells; epithelial sodium channel; single-channel recording; Erythrina crystagalli lectin; dopamine D1 and D2 receptors

PRECISE REGULATION OF ALVEOLAR surface fluid is critical for optimal gas exchange and is achieved through vectorial transport of solutes across the alveolar epithelium from the lumen of alveoli and interstitial spaces. The traditional view of lung fluid clearance is that active Na+ reabsorption by alveolar epithelial cells creates the driving force used to clear fluids from the air space. The alveolar epithelium, composed of two morphologically distinct types of cells, referred to as type I (AT1) and type II (AT2), is connected by tight junctions and provides an effective barrier against leakage of interstitial contents into the alveolar spaces. AT2 cells, covering only 2–5% of the alveolar surface, are cuboidal and are typically located at the corners of alveoli. AT1 cells are terminally differentiated squamous cells, covering >95% of the peripheral lung. The role of AT2 cells in surfactant production and ion transport has been extensively investigated (12, 13, 19, 29). However, the general functions of AT1 cells, including their role in alveolar ion/fluid transport, remain unclear.

There is evidence for epithelial Na+ channels (ENaC) in AT1 cells. ENaC are members of the degenerin family of extracellular ligand-gated channels. Specifically, ENaC is a heteromultimeric channel composed of homologous {alpha}-, beta-, and {gamma}-subunits (8). Recently, using enzymatic digestion and mechanical separation of AT1 cells from adult rat lungs, Johnson et al. (20) showed that AT1 cells take up Na+ in an amiloride-inhibitable fashion and that these cells express proteins for all three ENaC subunits. In addition, Borok et al. (5) immunohistochemically identified {alpha}-ENaC and amplified {alpha}-ENaC message from AT1 cells. Under normal circumstances, ENaC is the rate-limiting step for normal Na+ reabsorption in epithelial cells, and ENaC activity controls alveolar salt and fluid absorption in the alveoli. These findings are underscored by the observation that transgenic {alpha}-ENaC-knockout mice die within 40 h of birth because of an inability to clear lung fluid (14, 18). The conventional wisdom has been that only AT2 cells transport Na+, but, because AT1 and AT2 cells express ENaC, both cell types might be responsible for Na+ transport and maintenance of air space fluid homeostasis. We have shown functional ENaC in AT1 cells that were isolated by enzymatic digestion and immunoaffinity purification, but we were concerned that the isolation procedure might alter the properties and prevalence of ion channels. It was also sometimes difficult to unequivocally determine the apical surface of the cells. However, in this study, we examined the specific channel characteristics of ENaC in a lung slice tissue preparation, circumventing concerns that the AT1 cell membrane might be damaged during the isolation procedure and confirming the presence in AT1 cells of highly Na+-selective and -nonselective cation (HSC and NSC, respectively) channels, which are blocked by amiloride and activated by dopamine, in an environment similar to that of AT1 cells in the lung.

Catecholamines can increase lung fluid clearance. Dopamine has been reported to increase Na+ channel and Na+-K+-ATPase activity in the alveolar epithelium to facilitate lung edema clearance (2–4, 10). The effects of dopamine appear to be mediated via the apically located dopamine D1 receptor and the basolateral dopamine D2 receptor. These two receptors exert their biological actions by coupling to and activating different G protein complexes. The D1 receptor interacts with the Gs complex to activate adenylyl cyclase, whereas the D2 receptor interacts with Gi or Gq to inhibit cAMP production or activate phospholipase C (7). We recently observed in L2 cells (a rat AT2 cell line) that activation of D1 receptors was responsible for increasing ENaC activity in cell-attached patches via a cAMP-mediated signaling pathway when dopamine was applied to the apical surface of cells (16); therefore, we were interested in whether AT1 cells also respond to dopamine. Thus we examined the effect of dopamine on Na+ channel activity in AT1 cells and whether D1 or D2 receptors might be involved in putative dopamine-induced effects.

Here, we show, for the first time, single-channel recordings of amiloride-sensitive ENaC from AT1 cells in lung slice preparations. We also show a dopamine-induced increase in ENaC activity in AT1 cells via D1 receptor activation. These findings provide evidence of a definitive role for AT1 cells in maintaining fluid/ion homeostasis in the alveolar spaces and a responsiveness of AT1 cells to catecholamine treatment.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lung tissue preparation. Male Sprague-Dawley rats were maintained with access to standard rat diet and water ad libitum. At 8–12 wk of age, the animals were anesthetized and killed in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. All animal protocols conform to National Institutes of Health animal care and use guidelines and were approved by Emory University, IACUC. After lung perfusion via the pulmonary artery with 75 ml of warm (35°C) PBS, warm 2% low-melting-point agarose in PBS was intratracheally instilled into the lungs to expand the air spaces and provide support for the tissue during the slicing process. Excised lungs were removed en bloc and chilled in cold PBS (4°C) to solidify the agarose, and a small block of tissue was separated from the largest lobe of the lung and mounted using surgical-grade cyanoacrylate adhesive onto a Vibratome (model VT1000S, Leica Microsystems). The Vibratome blade was set to high-frequency vibration but was advanced slowly through the continuously chilled tissue to make 250- to 300-µm tissue slices. The tissue samples were placed in 50:50 ice-cold DMEM-F-12 (containing 10% FBS, 2 mM L-glutamine, 1 µM dextrose, 84 µM gentamicin, and 20 U/ml penicillin-streptomycin) until patch-clamp analysis. Lung cells were treated and patched within 4 h of the initial tissue preparation.

Single-channel patch-clamp analysis of Na+ channels in AT1 cells. Lung slices were rinsed three times in recording solution (see below) before the patch-clamp procedure. The experiments were conducted at 22–23°C; all patch solutions for the cell-attached configuration contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES at pH 7.4. Gigaohm seals were formed using a fire-polished glass microelectrode (backfilled with patch solution), and the lung slices were immersed in patch solution. An amplifier (Axopatch 1-D, Molecular Devices), interfaced through an analog-to-digital board to a personal computer, collected single-channel data. Channel currents were amplified at 5 kHz and filtered at 1 kHz with a low-pass Bessel filter.

For cell-attached patches, voltages are given as the negative of the patch pipette potential (–Vp), which is the displacement of the patch potential from the resting potential. Positive potentials represent depolarizations, and negative potentials represent hyperpolarizations of the cell membrane away from the resting potential. We used the product of the number of channels (N) and the single-channel open probability (Po) as a measure of ENaC activity within a patch, as described in previously (9). NPo was calculated using FETCHAN and pCLAMP 6 software (Molecular Devices).

Chemicals. Dopamine (3-hydroxytyramine, hydrochloride) was purchased from Calbiochem. Amiloride hydrochloride hydrate, sulpiride, and SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-H-3-benzazepine hydrochloride] were purchased from Sigma. Dopamine and amiloride were soluble in patch-clamp solution and prepared immediately before use. Dopamine receptor antagonists (sulpiride and SCH-23390) were dissolved in ethanol at 0.01 M stock concentrations. The specified amounts of dopamine and receptor blockers were applied directly to the bath solution, whereas amiloride was backfilled into patch pipettes (see below).

Fluorescent microscopy. Fluorescein-labeled lectin from the cry-baby tree Erythrina crystagalli (ECL; Vector Laboratories) can be used as a vital stain for AT1 cells in 250- to 300-µm lung slice preparations. When used to label AT1 cells for the purpose of recording single-channel activity, ECL was diluted 1:1,000 directly into the patch solution in which the lung tissue was immersed. For immunohistochemistry, lung slices were fixed with 4% paraformaldehyde (10 min at room temperature) and then labeled with a 1:1,000 dilution (in PBS-BSA with 3% goat serum) of fluorescein-labeled ECL and/or with antibodies. Lung tissue slices were colabeled with ECL and a final 1:20,000 dilution of anti-surfactant protein A antibody (Chemicon) for 1 h to identify AT1 and AT2 cells, respectively. Lung slices were then incubated in goat anti-rabbit secondary antibody conjugated to Alexa 568 (1:20,000 dilution; Molecular Probes) for 30 min. D1 and D2 receptor antibodies (Chemicon) were used at a 1:1,000 dilution. Goat anti-rabbit antibody conjugated to Alexa 488 (Molecular Probes) was used as the secondary detection antibody for dopamine receptors at a 1:10,000 dilution for 1 h. All tissue samples were mounted onto a glass slide with ProLong antifade reagent (Molecular Probes) and imaged using a laser scanning confocal microscope (model 510 NLO META, Zeiss).

Methods for statistical analysis. Statistical significance between two groups was determined by paired or unpaired t-tests, as appropriate. When the comparison between more than one group was required, statistical significance was determined by one-way ANOVA. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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F-ECL identifies AT1 cells. ECL exhibits high binding specificity toward galactose residues on cell membrane surfaces. The binding patterns of lectins to rat lung alveolar epithelial cells have been characterized using postembedding cytochemical techniques (33), which showed that ECL reacted selectively with AT1 cells. As shown in Fig. 1, F-ECL can be used to identify AT1 cells in lung slice preparations.


Figure 1
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  		Fig. 1. Fluorescein-labeled Erythrina crystagalli lectin (F-ECL)-positive alveolar type I (AT1) cells. Paraformaldehyde-fixed 275-µm rat lung tissue slices were colabeled with F-ECL and surfactant protein A (SPA) with binding specificity for AT1 and alveolar type II (AT2) cells, respectively. A: differential interference contrast image of lung slice. Original magnification x40. B: round AT2 cells labeled with anti-SPA antibody and secondary goat anti-rabbit secondary antibody conjugated to Alexa red (568-nm excitation). C: flask-shaped AT1 cells labeled with F-ECL (488-nm excitation). D: merged images of labeled AT2 and AT1 cells (B and C) showing cells near the accessible surface of the slice. LSM 5 Image Browser software (Zeiss) was used to process images.

 
Although the data are not shown, we independently verified the binding specificity of F-ECL to AT1 cells. When the same lectin-labeling protocol was used, F-ECL did not bind to a proliferative AT2 cell line (rat L-2 cells). F-ECL positively labeled R3/1 cells [a newly characterized rat AT1-like cell line (24)], as well as AT1 cells in mixed populations of AT1 and AT2 cells obtained from enzyme digestion of lung epithelium. In the mixed-population study, the larger AT1 cells were lectin positive, whereas the smaller round AT2 cells were negative for F-ECL. The specificity with which F-ECL labels AT1 cells, coupled with the innate morphological differences between AT1 and AT2 cells, enabled us to identify AT1 cells for in situ patch-clamp studies.

AT1 cells have ENaC. Using F-ECL, we were able to positively identify AT1 cells for patch-clamp analysis, whereby we observed two types of Na+ channels with conductances and current-voltage relations similar to those of ENaC in other epithelial cells, including AT2 cells (9, 27). Seemingly, AT1 cells express HSC and NSC channels. Figure 2A is a representative recording from an AT1 cell with HSC and NSC channels within a very small surface area of patched membrane. In Fig. 2A, voltage values indicate displacement of the patch membrane potential away from the resting membrane potential (–Vp) of the cell. The point amplitude histogram (Fig. 2B) clearly shows that the cell membrane has HSC channels with small unitary currents of –0.22 pA, as well as NSC channels with larger unitary currents of –0.77 pA. Additionally, Fig. 2B shows that the open probability of these channels is very high; HSC and NSC channels spend very little time in the closed state, and multiple channels are open simultaneously. Indeed, the additional levels of channel openings (peaks) in Fig. 2B correspond to various summations of HSC (–0.22 pA) and NSC (–0.77 pA) currents from simultaneous openings. Current-voltage relations for HSC and NSC channels are shown in Fig. 2, C and D, respectively. In this recording, conductance of the HSC channels was ~5.5 ± 0.54 pS (Fig. 2C), with an apparent reversal potential positive to the apical membrane potential. The second type of channel in Fig. 2A had an average conductance of 17.6 ± 1.25 pS and a linear current-voltage relation, characteristic of NSC channels, as described elsewhere (28). Interestingly, NSC channels in AT1 cells appear to have a reversal potential approximately –10 mV more negative than the apical membrane potential (Fig. 2D). This may be an important difference between AT1 and AT2 cells, in which NSC channel currents reverse at a potential more positive than the apical membrane potential. In Fig. 2E, we show the distribution of single-channel conductances from several different patches, in which we obtained currents at four or more holding potentials (to accurately calculate slope conductance). The mean conductance of AT1 cell HSC channels is 8.2 ± 2.5 pS and that of NSC channels is 22 ± 3.2 pS.


Figure 2
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  		Fig. 2. Single Na+ channel characteristics in AT1 cells. A: cell-attached patch of a lectin-positive, flask-shaped AT1 cell accessed from a live lung tissue slice with 2 distinct conductances. Arrows indicate closed state of the channel, downward deflections represent inward current, and upward deflections from the closed state represent outward current. Typical highly Na+-sensitive cation (HSC) channel openings are enclosed in boxes, and nonselective cation (NSC) channel activity is highlighted gray. B: point amplitude histogram from cell-attached patch in A at +40 mV and 30-s recording time. Unitary currents of HSC and NSC channels are shown; multiple peaks represent simultaneous channel openings. C: current-voltage (I-V) relation for AT1 cell HSC channels. Values were obtained from current events from patch-clamp recording in A. Calculated HSC channel conductance (G) is 5.5 ± 0.54 pS. D: I-V relation of NSC channels in AT1 cell with conductance of 17.6 ± 1.25 pS; values were obtained from current events from patch-clamp recording in A. E: distribution of single-channel conductances in AT1 cells accessed from lung slice tissue preparations.

 
Amiloride sensitivity of Na+ channels in AT1 cells. To test whether Na+ channels in AT1 cells are sensitive to low concentrations of amiloride, the tips of the pipettes were initially filled with regular patch-clamp solution, and the remainder of the pipette was backfilled with the same solution containing 25 nM amiloride. We previously calculated the approximate diffusion time for amiloride within the pipette (23). In a typical experiment, ~10 min are required for amiloride to reach the surface of the cell in a pipette tip used for single-channel patch-clamp analysis; this allows a patch to be used as its own control. Indeed, before complete amiloride blockade of Na+ channels (at 10 min) in Fig. 3 (at 0 mV applied potential), Na+ channel activity can be seen in the patch recording. However, after another ~1 min of amiloride diffusion, Na+ channel activity in AT1 cells was completely blocked, even at hyperpolarizing potentials of –20 and –40 mV (–Vp). Interestingly, at more negative intracellular potentials (–Vp = –60 and –80 mV), some Na+ channel activity could still be detected (albeit with smaller unit currents than were initially observed at 10 min or than would be expected at such large hyperpolarizing potentials). This was expected, because the amiloride concentration is less than the half-blocking concentration of amiloride of ~48 nM (19). By initially filling tips with regular patch solution before backfilling pipettes with 25 nM amiloride, in combination with applying large hyperpolarizing potentials, we are confident that the patches of membrane we examined indeed had ENaC activity initially. Therefore, this approach was taken in all patches used to examine the efficacy of amiloride inhibition. A summary of the results obtained at –40 mV (–Vp) displacement potentials is shown in Fig. 3B; average ENaC NPo from four independent AT1 cell recordings decreased from 0.481 ± 0.126 to 0.0186 ± 0.003 (P < 0.05). Higher amiloride concentration (50 nM) completely blocked ENaC function at all holding potentials (data not shown).


Figure 3
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  		Fig. 3. Amiloride inhibition of Na channels in AT1 cells. A: tip of patch-clamp electrodes was filled with regular patch solution and then backfilled with 25 nM amiloride. Na+ channel openings were present before complete amiloride diffusion to electrode tip (10 min) and appeared again at strongly polarized potentials of –60 and –80 mV (–Vp). Arrows indicate channel closed state, and downward deflections represent channel openings. B: epithelial Na+ channel (ENaC) NPo [product of number of channels (N) and single-channel open probability (P)] data from 4 independent patch studies show that 25 nM amiloride significantly inhibits Na+ transport at –40 mV (–Vp) from 0.481 ± 0.126 (control) to 0.0186 ± 0.003 (amiloride). *P < 0.05.

 
Dopamine increases ENaC NPo in AT1 cells. Dopamine stimulates lung liquid clearance, especially when edema accompanies lung injury. Classically, dopamine exerts its effects by binding to the D1 or the D2 receptor (22). The response of lung fluid clearance to dopamine and dopamine receptors has been investigated only in AT2 cells or in whole lung perfusion studies. Because most of the alveolar surface consists of the apical membranes of AT1 cells, it is important to examine dopamine receptor expression and function in AT1 cells.

We began by investigating whether AT1 cells express detectable levels of dopamine receptors on the cell surface. Confocal images in Fig. 4A show D2 and D1 receptor expression in 250-µm lung slices fixed in paraformaldehyde. In both images, the characteristic morphology of AT1 cells (Fig. 1) was clearly visible in cells labeled with dopamine receptor antibodies. This strongly suggested that dopamine receptor subtypes are present in AT1 cells and that AT1 cells should be responsive to catecholamine treatment.


Figure 4
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  		Fig. 4. Dopamine receptor expression and activation in AT1 cells activate ENaC function. A: confocal images of dopamine (D2 and D1) receptor antibody binding in paraformaldehyde-fixed 250-µm rat lung slice preparations. B: after control ENaC activity was obtained, 10 µM or 3 mM dopamine added to bath solution significantly increased Na+ channel activity. ENaC NPo increased from 0.31 ± 0.19 to 0.60 ± 0.21 (n = 10, P = 0.005) with 10 µM dopamine and from 0.38 ± 0.17 to 1.2 ± 0.32 (n = 11, P = 0.001) with 3 mM dopamine. C: representative cell-attached patch recording from an AT1 cell accessed from a lung slice preparation treated with 10 µM and then with 3 mM dopamine, with application of 0-mV potential.

 
Then we applied dopamine to the external patch solution of single-channel recordings to examine the effect of catecholamine treatment on ENaC NPo. In these experiments, we formed a cell-attached patch and then recorded control activity for several minutes before applying dopamine to the bath solution. In this way, each cell patch served as its own control, and statistical significance was determined by paired t-test. ENaC NPo before and after dopamine is plotted for each cell in Fig. 4B. Average NPo values significantly increased from 0.31 ± 0.19 to 0.60 ± 0.21 (n = 10) in the presence of 10 µM dopamine (P = 0.005). Our studies also included higher concentrations of dopamine (up to 3 mM). High concentrations of dopamine continued to increase ENaC NPo significantly from 0.38 ± 0.17 to 1.2 ± 0.32 (n = 11, P = 0.001; Fig. 4B). Finally, in Fig. 4C, we show a representative recording from a cell that was first treated with vehicle (saline solution), then with 10 µM dopamine, and finally with 3 mM dopamine. In this patch, we were able to maintain a tight seal on an AT1 cell membrane for an extended period, which permitted sequential application of several concentrations of dopamine. Although not explicitly examined in our studies, the number of channels per patch (N) appeared to be increased after 10 µM and 3 mM dopamine (Fig. 4C), because two open states appear more often than before catecholamine treatment.

D1 receptor antagonist SCH-23390 blocks the effect of dopamine on ENaC. Because D1 and D2 receptors were present in lung tissue, we next wanted to determine which receptor type was mediating the dopamine-induced increase in ENaC activity. We found that application of the D1 receptor blocker SCH-23390 (10 µM) to AT1 cells after seal formation blocked the effect of 10 µM dopamine on ENaC NPo. However, the D2 receptor blocker sulpiride (10 µM) did not inhibit dopamine activation of Na+ channel activity. [We also found that sulpiride and SCH-2390 inhibitors alone did not significantly change Na+ channel transport from control levels (data not shown).] Figure 5A shows that ENaC NPo increased significantly from 0.64 ± 0.16 to 1.25 ± 0.27 after 10 µM dopamine, even in the presence of the D2 receptor antagonist, whereas the D1 receptor blocker prevented the established dopamine-induced increases in ENaC NPo (Fig. 5B).


Figure 5
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  		Fig. 5. D1 receptor antagonist SCH-23390 blocks effect of dopamine on ENaC. A: D2 receptor antagonist sulpiride (10 µM) failed to block 10 µM dopamine activation of ENaC NPo. B: D1 receptor blocker SCH-23390 (10 µM) blocked effect of dopamine on ENaC NPo. C and D: representative recordings for 10 µM sulpiride and 10 µM SCH-23390, respectively. In both cases, after a control recording period, receptor antagonists and then 10 µM dopamine were applied to the bath solution. Sulpiride or SCH-23390 alone did not significantly change channel activity; single-channel activity remained unchanged. #, Break in continual single-channel recordings; effect of dopamine in the presence of sulpiride or SCH-23390 was evaluated after 5 min of dopamine application. After treatment with sulpiride, but not SCH-23390, dopamine continued to increase open probability of the channels.

 
Representative/typical single-channel recordings are shown in Fig. 5, C and D. Approximately 1 min of the control recording period was followed by application of D2 or D1 receptor blocker. For 2 min after addition of the receptor inhibitor, we found that dopamine receptor antagonists (alone) did not alter Na+ channel properties in AT1 cells. At 3 min, we subjected the same cell that had been treated with D2 or D1 receptor blocker to a 10-µM dopamine challenge. At ~5 min after the initial application of dopamine, we evaluated the effect of receptor inhibition and dopamine application on ENaC NPo. In the presence of the D2 receptor inhibitor, dopamine increased ENaC activity within minutes (Fig. 5C). A second open level of Na+ channels is very apparent in the recording period following sulpiride and dopamine treatment (similar to dopamine only in Fig. 4C). Conversely, dopamine did not increase ENaC NPo in AT1 cells treated with the D1 receptor blocker. Single-channel current remained unchanged (Fig. 5D). These studies strongly suggest that the effect of dopamine on ENaC is mediated through D1 receptors.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Role of AT1 cells in alveolar solute transport. The traditional view of lung fluid clearance is that active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from air spaces. Although ion transport in AT2 cells has been extensively studied (13, 19, 29), the contribution of AT1 cells to alveolar solute transport is not clearly understood.

Our study examined the transport properties of AT1 cells accessed from 250- to 300-µm sections of live lung tissue. Studying AT1 cells in situ obviates concerns that protracted cell isolation procedures might compromise the properties of native lung cells.

Using a similar technique, Bourke et al. (6) also made patch recordings from live tissue preparations and demonstrated that instillation of agarose followed by sectioning of lung slices allows examination of AT1 cells. They found that AT1 cell viability in slice preparations can be maintained for several hours after the surgical procedure. In our studies, we have further shown that this new procedure for accessing AT1 cells in live tissue does not alter epithelial cell transport properties and that F-ECL can be used as a vital stain for AT1 cells. The present study provides, for the first time, unequivocal evidence for the presence of ENaC in native AT1 cells and raises the possibility that the entire alveolar epithelium (composed of AT1 and AT2 cells) participates in alveolar fluid homeostasis. Prior to our present study, Bourke et al. (6) successfully recorded ion channel activity in a lung slice preparation. However, they were unable to record ENaC activity; instead, they reported, in the presence of many inhibitors, a 21-pS voltage-dependent channel with characteristics consistent with K+ channels. We found that the biophysical properties of Na+-permeant channels in membrane patches from AT1 lung slice preparations are similar to those of HSC, as well as NSC, channels that have been reported in AT2 cells (19). HSC and NSC channels in AT1 cells were occasionally coexpressed in very small (<-1 µm) patches of the cell membrane and spent very little time in the closed state (Fig. 2). In our present studies, we were able to form seals in >90% of all lung slice tissue preparations obtained from healthy, well-perfused rat lungs; from these, ENaC activity was recorded 50% of the time. This suggests to us that ENaC is abundant in the apical membrane of AT1 cells in the lung slice preparation. This assumption is consistent with the finding that the reversal potentials of NSC channels from AT1 cells are slightly hyperpolarized. The –10-mV shift of the reversal potential (compared with NSC reversal potentials near 0 mV in AT2 cells) provides evidence that AT1 and AT2 cells play different roles in regulating alveolar fluid homeostasis and that Na+ channels may be more abundant in AT1 than in AT2 cells in the apical membrane. Johnson et al. (20) reported that Na+ uptake per microgram of protein was 2.5 times greater in AT1 than in AT2 cells. Together, these studies show that AT1 cells indeed play an important role in maintaining alveolar ion homeostasis, which affects total alveolar fluid balance.

Dopamine increases lung fluid clearance. Because catecholamines play an important role in lung liquid removal in the adult and newborn lung, we examined the responsiveness of ENaC in AT1 cells to dopamine. In humans, dopamine concentrations in the lungs can be very high, above levels reported in the adrenal glands (17), and circulating levels of dopamine sulfate can increase dramatically postprandially (15). In late-term fetal sheep, the average plasma level of dopamine rises 10-fold immediately before delivery (21, 26, 31). Furthermore, in several species, the fully developed and active pulmonary neuroendocrine system at birth contributes to local surges of catecholamine production (11). These studies suggest that dopamine is important in lung liquid removal at birth. Indeed, several isolated perfused lung studies have shown that dopamine substantially increases liquid clearance in lung epithelium (3, 4, 10). Despite these studies, the mechanism by which dopamine regulates fluid reabsorption remains unclear.

We have demonstrated that dopamine increases ENaC activity in AT1 (present study) and AT2 (16) cells. Therefore, we conclude from our studies that catecholamines can improve lung fluid clearance by increasing amiloride-sensitive ENaC activity in cells that comprise the entire alveolar epithelium. However, two independent studies showed that dopamine stimulation of Na+ transport and fluid clearance in the lung epithelium does not involve amiloride-sensitive channels (4, 10). In each study, 10–6–10–4 M amiloride did not block dopamine stimulation of lung fluid clearance. Although it is possible for dopamine to affect amiloride-insensitive Na+ channels (which have been reported in the alveolar epithelium), our present study demonstrates a definite role for dopamine regulation of ENaC in AT1 cells and is at variance with these previously published reports.

Our present report and several previous studies in lung epithelia (1, 16, 25, 30, 32) suggest that dopamine binds to and activates D1 receptors to increase lung liquid clearance. In Fig. 5A, we clearly show that Na+ channel activity, which is responsible for the balance of reabsorption and secretion that maintains a closely controlled level of alveolar fluid, fails to respond to dopamine in the presence of a specific D1 receptor blocker. Similar to our present findings and our recent study of the effect of dopamine on AT2 cells (16), Adir et al. (1, 2) also reported that D1, but not D2, receptor antagonists inhibited the ability of endogenously produced dopamine to increase lung liquid clearance in lung cells. However, we believe that the effect of dopamine on AT1 D1 receptors differs from D1 receptor-mediated effects in AT2 cells. In this study, we have shown that dopamine does not increase the open probability of ENaC channels, as it does in AT2 cells (16); rather, dopamine might increase the number of channels in a patch (Fig. 4C). Apparently, D1 receptors are linked to different signaling cascades in AT1 cells, which requires further investigation.

In summary, we have developed a lung slice model for studying AT1 cells in an environment that is close to that of lung cells. Our use of F-ECL as a vital stain for identifying ATI cells in a lung slice preparation allowed us to positively identify AT1 cells in situ for single-channel patch-clamp analysis of cells. This led to the identification of amiloride-sensitive HSC and NSC channels, with mean conductances of 8.2 ± 2.5 and 22 ± 3.2 pS, respectively, in the membrane of AT1 cells. We also found that AT1 cells have D1 and D2 receptors and that dopamine acts through D1 receptors to increase net Na+ transport in alveolar epithelium. Overall, it is becoming increasingly apparent that AT1 cells contribute significantly to lung fluid homeostasis.


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 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R24 DK-064399 to D. C. Eaton, T32 AA-013528 to M. N. Helms, and R01 HL-071621, R01 HL-063306, and P50 AA-013757 to D. C. Eaton and L. C. Job.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Roland Koslowski (Dresden University of Technology, Dresden, Germany) for providing R3/1 cells.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Eaton, Dept. of Physiology, Emory Univ. School of Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322 (e-mail: deaton{at}emory.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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