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Am J Physiol Lung Cell Mol Physiol 290: L710-L722, 2006. First published November 11, 2005; doi:10.1152/ajplung.00486.2004
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Dopamine regulation of amiloride-sensitive sodium channels in lung cells

My N. Helms,2,3 Xi-Juan Chen,1,3 Semra Ramosevac,1,2,3 Douglas C. Eaton,1,2,3 and Lucky Jain1,2,3

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

Submitted 30 December 2004 ; accepted in final form 3 November 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dopamine increases lung fluid clearance. This is partly due to activation of basolateral Na-K-ATPase. However, activation of Na-K-ATPase by itself is unlikely to produce large changes in transepithelial transport. Therefore, we examined apical and basolateral dopamine's effect on apical, highly selective sodium channels [epithelial sodium channels (ENaC)] in monolayers of an alveolar type 2 cell line (L2). Dopamine increased channel open probability (Po) without changing the unitary current. The D1 receptor blocker SCH-23390 blocked the dopamine effect, but the D2 receptor blocker sulpiride did not. The dopamine-mediated increase in ENaC activity was not a secondary effect of dopamine stimulation of Na-K-ATPase, since ouabain applied to the basolateral surface to block the activity of Na-K-ATPase did not alter dopamine-mediated ENaC activity. Protein kinase A (PKA) was not responsible for dopamine's effect since a PKA inhibitor, H89, did not reduce dopamine's effect. However, cpt-2-O-Me-cAMP, which selectively binds and activates EPAC (exchange protein activated by cAMP) but not PKA, increased ENaC Po. An Src inhibitor, PP2, and the phosphatidylinositol-3-kinase inhibitor, LY-294002, blocked dopamine's effect on ENaC. In addition, an MEK blocker, U0126, an inhibitor of phospholipase A2, and a protein phosphatase inhibitor also blocked the effect of dopamine on ENaC Po. Finally, since the cAMP-EPAC-Rap1 pathway also activates DARPP32 (32-kDa dopamine response protein phosphatase), we confirmed that dopamine phosphorylates DARPP32, and okadaic acid, which blocks phosphatases (DARPP32), also blocks dopamine's effect. In summary, dopamine increases ENaC activity by a cAMP-mediated alternative signaling pathway involving EPAC and Rap1, signaling molecules usually associated with growth-factor-activated receptors.

single channel recording; ion transport; epithelial sodium channels; L2 lung cells; exchange protein activated by cAMP

EXCESSIVE ACCUMULATION of fluid in the alveolar spaces frequently accompanies acute lung injury (87), and the failure of the lungs to rapidly clear this edema fluid has been related to higher morbidity and mortality (51). Because alveolar fluid clearance is driven by active transport of Na and water across the epithelial lining of air spaces, investigative efforts have focused on strategies to enhance this process (71, 83). There is general agreement that Na transport across Na-reabsorbing tight epithelia such as the lung alveolus occurs via a two-step process (50). First, Na enters alveolar epithelial cells from the surface lining fluid through Na permeability pathways in the apical membrane, before it is actively transported out of the cell by the basolateral Na-K-ATPase. A significant fraction of the net alveolar Na absorption can be inhibited by amiloride, and since molecular biological studies have confirmed the presence in lung epithelia of the three epithelial Na channel subunits, {alpha}, beta, and {gamma}, it has generally been assumed that a large part of lung Na transport is mediated by some form of amiloride-sensitive epithelial Na channels (ENaC). Because maintaining an appropriate fluid layer is critical for normal lung function, the transepithelial Na transport rate in lung epithelium must be subject to very close regulation. In theory, regulation of Na transport could occur at the exit step by controlling the activity of the Na-K-ATPase or through modulation of the entry step, i.e., the Na channel. In fact, there is substantial evidence that both steps are regulated in a physiologically relevant way to control fluid levels on the surface of alveoli. This regulation is in contrast to other Na-transporting epithelia like the kidney and the colon, where the entry step appears to be the primary control point and there is enormous excess capacity of the Na-K-ATPase to transport Na.

Dopamine has been shown to increase lung liquid clearance under basal conditions (6, 7) and in situations where edema accompanies lung injury (69, 70). Barnard et al. (7) reported that stimulation of D1 dopamine receptors on the basolateral surface of alveolar type II epithelial cells (AT2 cells) resulted in activation of Na-K-ATPase. Despite this observation, there are also reports that some increase in lung fluid clearance is mediated by D1 activation of apical Na channels (1). We thought that this effect might be through a direct effect of dopamine on apical or basolateral D1 receptors activating apical ENaC in AT2 cells. In our work, we have examined the effect of dopamine on highly selective Na channels (ENaC) in a rat epithelial cell line (L2), which is a good model for AT2 cells. We also explored the possible targeting of dopaminergic receptors and the signaling pathways for this dopamine effect. This work demonstrates a cAMP-mediated effect of dopamine on Na channels that is consistent with a coordinated response of Na-K-ATPase and Na channels to promote maximal increases in transport and alveolar fluid clearance. The cAMP effect is interesting in that it is not mediated by protein kinase A (PKA), but rather by an alternative pathway that involves signaling molecules usually associated with growth factor-activated receptors.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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L2 cells, originally from AT2 cells from adult rat lung (17), were obtained from American Type Culture Collection (ATCC, Rockville, MD; CCL-149) and maintained using standard cell culture methods. L2 cells are cultured in Ham's F-12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate with 10% fetal bovine serum at a temperature of 37°C. The medium was replaced two to three times per week. We transferred the cells to collagen-coated permeable supports with an air interface until they reached confluence before using the monolayers for patch-clamp experiments. Patch-clamp experiments were performed at room temperature as we have described in the previous papers (12, 34, 35). In brief, the pipettes were pulled from filamented borosilicate glass capillaries (TW-150F, World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). The pipettes were coated with Sylgard (World Precision), and tips were fire polished (Narishige). The resistance of these pipettes was 5–8 MW when filled with the pipette solution.

We used the cell-attached configuration for most of our studies since, in this case, the cytoplasmic constituents remain intact, thus allowing us to study the role of cytoplasmic second messengers in the regulation of ion channel activity. In addition, cell-attached patches can be perfused after a control recording period with the various drug agents used in this study. Thus patches with channel activity can serve as their own controls, which allows us to apply paired statistics and, therefore, eliminates the statistical problems with the wide variability in open probability (Po) seen in control patches. The bath and pipette solutions used in the cell-attached mode contained (in mM) 140 NaCl, 1 CaCl2, 5 KCl, and 10 HEPES, pH adjusted to 7.4 with 2 N NaOH. After formation of a high-resistance seal (>50 GW), the channel currents were recorded with an Axopatch 200A amplifier (Axon Instruments, CA) with a low-pass 1-kHz Bessel filter. The channel activity was sampled at 5 kHz by a computer with pCLAMP 6 software (Axon Instruments, CA). The channel Po and unitary current were measured from the stable, continuously recorded data using the FETCHAN program in pCLAMP 6. Unless specified otherwise, each cell-attached patch served as its own control. Dopamine and other agents being studied were added to the media covering the apical or basolateral surface as needed in a specially designed chamber to optimize patch efficiency and allow separate control of apical and basolateral medium (Fig. 1). We have previously published details of this experimental protocol (12). The single channel conductance was determined by a linear regression of unitary current amplitudes over the range of pipette holding potentials. Statistical analysis for the changes in Po and unitary currents of the channels was performed with SigmaStat for Windows (Jandel Scientific). Statistical significance between two groups was determined by paired (for all measurements of single channel activity) or unpaired tests, as appropriate. When the comparison between more than one group was required, statistical significance was determined by one-way ANOVA or one-way repeated-measures ANOVA (for cell-attached patch measurements) followed by comparison of treated with untreated cells by Dunnett's test or pair-wise comparisons with the Holm-Sidak posttest to determine significant differences. P values <0.05 were considered significant.


Figure 1
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  		Fig. 1. Schematic diagram of the specialized chamber for recording single channel currents from L2 (and other lung epithelial) cells. This chamber is placed on the stage of an inverted microscope and allows substantial clearance between the cell surface and the microscope condenser.

 

   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Under appropriate conditions, L2 cells express epithelial Na channels that are indistinguishable from those observed in AT2 cells. L2 cells are a spontaneously transformed rat lung alveolar cell line available from ATCC (17). They have all of the phenotypic characteristics of AT2 cells (i.e., surfactant production and amiloride-sensitive transepithelial current) (75) and have been previously used by others as a model for AT2 cells (77, 82). They have not lost their contact inhibition like some lung cell lines derived from tumors (e.g., A549 cells) but do not form monolayers with as high a resistance as AT2 cells. Nonetheless, it is possible to measure transepithelial current that is sensitive to 1 µM amiloride (Fig. 2A). Moreover, when grown under conditions that we have found to promote expression of highly selective, low-conductance Na channels in AT2 cells (35) (permeable supports, an air interface, and steroid hormones), L2 cells also express ENaC. The biophysical properties of these channels are similar to those of the channels reported by us from AT2 cells (6 pS conductance, highly Na selective with a reversal potential >100 mV more depolarized than the resting potential in cell-attached patches, inhibited by submicromolar concentrations of amiloride) (35) (Fig. 2B). In particular, the single channel conductance is statistically the same. There is some variability in the Po (Fig. 2C) and mean open and closed times (closed time = 2.34 ± 0.106 s, open time = 0.734 ± 0.0668 s; means ± SE, n = 139). Mean open times are not distributed normally, with shorter mean open times favored over longer ones. But this variability is characteristic of ENaC recorded from many different Na-transporting epithelial cells, including AT2 cells (12, 22, 34, 35, 63). Therefore, L2 cells appear to be a useful model for examining ENaC in a continuous cell line.


Figure 2
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  		Fig. 2. Characteristics of L2 cells as a model for alveolar type II epithelial (AT2) cells. A: L2 cell monolayers have amiloride-sensitive transepithelial current. L2 cells were cultured on permeable supports as described in METHODS, transepithelial resistance and voltage were measured, and transepithelial current was calculated. In every case, 1 µM amiloride reduced the current, demonstrating that L2 cells are a good model in which to examine ENaC. B: current-voltage relationship for L2 cell highly selective Na channels. Values were obtained from current events from all patches on L2 cells prior to treatment with any agent (137 patches). The slope of the line is 6.7 pS between –60 and –20 mV. The very positive reversal potential (not resolved in this plot) shows that the channel is highly selective for Na+ over K+. C: distribution of open probabilities (Po). ENaC has a wide distribution of Po in L2 cells as in other epithelial preparation. The natural distribution of Po means that the relative open time of epithelial sodium channels (ENaC) can vary enormously from 1 patch to the next (compare the records in Figs. 3 and 10).

 
Dopamine increases the activity of ENaC. When applied to the apical surface of L2 cells, dopamine (10 µM) produces a consistent increase in the Po of ENaC without significantly changing the unitary current of the channel (Fig. 3, A and B, left). Despite the variability in baseline Po, the channels recorded in these patches were all HSC with similar single channel currents and current-voltage relationships to those shown in Fig. 2. Interestingly, dopamine also produces a comparable increase in Po when the same concentration is applied to the basolateral surface (Fig. 3B, right). There was no apparent increase in number of channels per patch, implying that the major effect of dopamine on ENaC activity was to alter Po rather than N, the density of Na channels. To explicitly examine this question, we measured the amount of ENaC subunit protein that was subject to biotinylation and was, therefore, available on the surface of the cell. Application of apical dopamine does not substantially change the amount of any ENaC subunit in the apical membrane (Fig. 4).


Figure 3
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  		Fig. 3. Dopamine (10 µM) increases Po of ENaC. A: typical single channel records from a L2 patch before and after application of dopamine (10 µM) to the apical surface of the cell. Dopamine increases the activity of the channels by increasing the Po and the mean open time of ENaC. c and o, Closed and open levels, respectively. The results from several patches are summarized in B for dopamine (10 µM) applied to the apical (left) or basolateral (right) side of L2 monolayers. Po after the treatment was significantly higher than control, but the unitary currents of the channels were not significantly changed (from 0.133 ± 0.036 to 0.142 ± 0.044 for the apical side and from 0.190 ± 0.051 to 0.192 ± 0.010 for the basolateral side). The bars represent SD.

 

Figure 4
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  		Fig. 4. Dopamine does not increase the amount of ENaC in the apical membrane. To determine whether dopamine promoted insertion of ENaC into the apical membrane, the apical membranes of L2 cells in the presence or absence of dopamine were biotin labeled, the cells were lysed, and all biotin-labeled proteins were precipitated with streptavidin. The biotin-labeled proteins were resolved on an SDS gel, blotted, and detected with ENaC subunit-specific antibodies. Dopamine does not change the amount of any of the subunits.

 
Both dopamine D1 and D2 receptors are present in L2 and AT2 cells. There are at least five different dopamine receptor isoforms; however, in whole lung and isolated AT2 cells only two, D1 and D2, are present in significant amounts (7, 14, 64). In Western blots of cell lysates from isolated L2 cells, we could detect both D1 and D2 receptors (Fig. 5, insets). In a further effort to validate L2 cells as a model for AT2 cells, we also examined, using antibodies from two different sources, whether AT2 cells also had both types of dopamine receptors. As we observed in L2 cells, AT2 cells contained both D1 and D2 receptors, although there appeared to be more D1 receptors than D2 receptors (Fig. 6).


Figure 5
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  		Fig. 5. Western blots in insets show that both D1 and D2 receptors are present, but D1 appears to be the most prevalent. The D1 receptor blocker SCH-23390 (10 µM) blocks the effect of dopamine (A); however, the D2 receptor blocker sulpiride (10 µM) does not block the effect of dopamine when applied to either side of a L2 monolayer (B). After a control recording period, SCH-23390 or sulpiride was applied to either the apical or basolateral side of a L2 monolayer for ~5 min. Then, 10 µM dopamine was applied to the same side of the monolayer. SCH-23390 or sulpiride alone did not significantly change channel activity. After the treatment with sulpiride but not SCH-23390, dopamine still increased the Po of the channels. The unitary current remained relatively unchanged regardless of treatment. Thus the effect of dopamine on highly selective channels (HSC) is apparently mediated through D1 dopamine receptors.

 

Figure 6
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  		Fig. 6. D1 and D2 receptors are also present in AT2 cells. To demonstrate the similarity of L2 cells to AT2 cells for the purposes of this paper, we isolated AT2 cells as we have done before and lysed the cells and in Western blots probed for D1 and D2 receptors with two different commercially available antibodies (A, Santa Cruz; B, Chemicon). In both cases, both receptors could be detected, but D1 receptors appeared more prevalent (as they were in L2 cells).

 
Only a D1 receptor antagonist blocks the effect of dopamine on ENaC. Because both receptor types were present in both cell types, we wished to determine which receptor type was mediating the dopamine-induced increase in ENaC Po. The D1 receptor blocker, SCH-23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-H-3-benzazepine hydrochloride, 10 µM] blocked the effect of dopamine on ENaC Po (Fig. 5A) when applied to either the apical or basolateral side of an L2 monolayer; however, the D2 receptor blocker, sulpiride (10 µM), did not block the effect of dopamine. In both cases, after a control recording period, the receptor antagonist was applied to either the apical or basolateral side of an L2 monolayer for ~5 min. Then, 10 µM dopamine was applied to the same side of the monolayer. SCH-23390 or sulpiride alone did not significantly change channel activity. The single channel current remained relatively unchanged regardless of treatment. Thus the effect of dopamine on ENaC is apparently mediated through D1 dopamine receptors.

The dopamine effect on ENaC is not due to a change in basolateral Na-K-ATPase activity. Several previous reports have documented that one action of dopamine is to increase the activity of Na-K-ATPase in lung epithelial cells. It is possible that dopamine activation of Na-K-ATPase could lead to changes in the intracellular environment that might alter ENaC activity independently of any direct activation of ENaC by dopamine. Therefore, we examined whether dopamine was still capable of activating ENaC even when Na-K-ATPase was inhibited. Patches were formed on cells, and single channels were recorded for ~10 min, after which 1 mM ouabain was added to the basolateral surface of the cells. Ouabain inhibits Na-K-ATPase rapidly, and this inhibition is reflected in an almost immediate reduction in the electrogenic current produced by the ATPase, but despite inhibition, there should be (as previously demonstrated in other Na-transporting epithelial cells many years ago) little initial change in the intracellular Na concentration of the cell (1 meq or less) over a period of 20 min after ouabain application, a change that would not be sufficient to cause cell swelling, or a downregulation of the ENaC channels (18, 19, 41). Of course, over a much longer period there will be substantial changes in the intracellular ion concentrations that will lead to profound changes in intracellular processes including cell volume, cell pH, and other properties, but these changes take tens of minutes or hours (18, 19, 41). Nonetheless, we can tell that exposure to ouabain blocks Na-K-ATPase rapidly, since the loss of the electrogenic activity that normally hyperpolarizes the apical membrane is reflected in an apical depolarization that decreases the unit current after application of ouabain (from 0.232 ± 0.0444 pA before ouabain to 0.176 ± 0.0401 pA after ouabain; means ± SD, n = 9). Despite the fact there is a clear inhibition of the Na pump, the effect of dopamine still produces a large increase in ENaC Po (Fig. 7). Thus the effect of dopamine on ENaC does not depend upon or require the activity of Na-K-ATPase even though dopamine may independently alter Na-K-ATPase activity.


Figure 7
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  		Fig. 7. Dopamine still increased Po of ENaC after Na-K-ATPase had been blocked by ouabain. Because dopamine does activate Na-K-ATPase, the effect on ENaC might be secondary to inhibition of the pump. After control HSC channel activity had been obtained, 1 mM ouabain was applied to the basolateral side of a L2 monolayer for ~5 min. The ENaC unitary current was decreased after the treatment of ouabain (reduced driving force), but Po was not significantly changed. However, when 10 µM dopamine was subsequently added to the apical side of the L2 monolayer, Po was significantly increased above the ouabain-induced level. Thus the dopamine effect on ENaC was not secondary to an effect on Na-K-ATPase.

 
The D1 receptor is linked to the G protein, Gs, not Gi. Dopamine receptors are G protein-linked receptors. In the literature, D1 receptors have been variously reported to associate with some Gs, Gq, Go, or Gi proteins either individually or in combination (54, 57, 76). We and others have previously shown that activation of Gq mostly by purinergic receptors uniformly and profoundly inhibits ENaC (33, 46). Therefore, it seemed unlikely that apical D1 receptors were linked to Gq. If the D1 receptor were linked to Gi or Go, then the dopamine-mediated stimulation of ENaC should be blocked by the Gi/o inhibitor, pertussis toxin (PTX) (25, 26). L2 cells were pretreated with 1 µg/ml PTX for 4 h; then patches were formed, and single channel activity was recorded from the same cell before and after treatment with 10 µM dopamine applied to the apical surface of the monolayers. Although PTX may have increased the initial Po (0.147 ± 0.123, mean ± SD, n = 8), dopamine still significantly increased Po (0.370 ± 0.139, means ± SD, n = 8; see Fig. 8A), suggesting that neither Gi nor Go was involved in the apical D1 receptor activation of ENaC. To test whether the receptors were linked to Gs, we pretreated the cells with cholera toxin (CTX), which prevents interaction of Gs with its receptors and persistently activates Gs (68, 73). L2 cells were pretreated with 0.1 µg/ml CTX for 4 h; then patches were formed, and single channel activity was recorded on the same cell before and after treatment with 10 µM dopamine applied to the apical surface of the monolayers. CTX did produce an increase in the number of ENaC per patch as has been reported in other Na-transporting epithelial cells (48); however, CTX blocked the ability of dopamine to increase ENaC Po (initial Po = 0.476 ± 0.126, after dopamine Po = 0.443 ± 0.161, means ± SD, n = 3; Fig. 8B), implying that D1 receptors did link to and activate Gs.


Figure 8
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  		Fig. 8. The effect of dopamine is not sensitive to pertussis toxin (PTX) but is sensitive to cholera toxin (CTX). L2 monolayers were pretreated with PTX (50 µg/ml), an inhibitor of Gi proteins, or CTX (0.1 µg/ml), an uncoupler of Gs proteins, for 4 h. In A, despite the treatment with PTX, 10 µM dopamine added to the apical side of the L2 monolayer still significantly increased ENaC Po. On the other hand, in B, treatment with CTX inhibited any increase in ENaC Po (although not shown, there was an increase in the number of channels per patch in response to CTX).

 
Dopamine increases cellular cAMP. One mechanism by which dopamine can produce its effect in some cell types is by activation of adenylyl cyclase. Such an activation is consistent with the D1 receptor acting through Gs. If adenylyl cyclase is activated there should be an increase in cellular cAMP. Figure 9A shows that dopamine does increase cAMP levels, but not as much as forskolin, a direct activator of adenylyl cyclase. The difference might be because D1 receptors only activate apical adenylyl cyclase, while forskolin activates adenylyl cyclase wherever it is in the cell.


Figure 9
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  		Fig. 9. Dopamine increases intracellular cAMP, but the PKA inhibitor H89 does not block the effect of dopamine. InA, cAMP was measured by a commercially available spectrophotometric binding assay. Dopamine significantly increased cAMP above baseline levels, but forskolin, a general activator of adenylyl cyclase, produced a substantially larger increase (*P < 0.05). In B, L2 cells were pretreated with 5 µM of the PKA inhibitor H89 for ~10 min, then the channel activity was recorded before and after treatment with 10 µM dopamine applied to the apical sides of the monolayers. Po was still significantly increased by dopamine.

 
PKA inhibitor H89 does not block the effect of dopamine. The typical result of the production of cAMP is activation of PKA (31). This possibility did not seem likely to us since in other work increased PKA activity does increase ENaC activity but does so by increasing the channel density (i.e., the number of channels per unit area of membrane) rather than, as dopamine does, by increasing ENaC Po (12, 48). Nonetheless, we examined the effect of inhibiting PKA on the dopamine response. L2 cells were pretreated with 5 µM of the PKA inhibitor H89 for ~30 min; then patches were formed, and single channel activity was recorded before and after treatment with 10 µM dopamine applied to the apical surface of the monolayers. As we had expected, Po was still significantly increased by dopamine (see Fig. 9B).

An alternative cAMP pathway activates ENaC. An alternative pathway by which dopamine can produce its effect is by activation of exchange protein activated by cAMP (EPAC). EPAC is the GTP exchange factor for a member of the Ras family of small G proteins, Rap1. Activation of EPAC promotes GTP binding to and activation of Rap1. In general, when cAMP increases, both PKA and EPAC are activated. It is possible to distinguish the effects of EPAC activation from that of PKA by using the membrane permeable cAMP analog, cpt-2-O-methyl-cAMP [8-(4-chlorophenylthio)-2'-O-methyl-cAMP], which binds to EPAC but does not bind or activate PKA (20, 36). Figure 10 shows that the EPAC activator increases ENaC Po just as dopamine does.


Figure 10
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  		Fig. 10. An activator of the exchange protein activated by cAMP (EPAC) mimics the action of dopamine. The membrane-permeable cAMP analog, 8-cpt-2'-O-methyl-cAMP, activates EPAC but does not activate PKA. When applied to L2 cells the analog increases ENaC Po like dopamine.

 
Dopamine-induced increases in ENaC activity require Src activity. Many G protein receptors, including D1 receptor, can activate cytosolic, nonreceptor tyrosine kinases that are members of the Src family of kinases (32). In fact, the EPAC-activated, small G protein Rap1 can be further activated by cytosolic tyrosine kinases of the Src family (60). Src itself is activated by cAMP (60, 72) so that the concerted action of EPAC and Src is often necessary to produce maximal activation of Rap1. Because of these previous observations, we examined whether inhibition of Src kinases could reduce the dopamine-induced increase in ENaC activity. We applied an inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) that blocks all members of the Src family of kinases. Interestingly, there appears to be a constitutive inhibition of ENaC associated with Src kinase activity since merely inhibiting Src family members alone produces a significant increase in ENaC activity (Fig. 11). However, after inhibition of Src kinases, dopamine was ineffective at increasing ENaC activity (Fig. 11A). Thus dopamine D1 receptor activation appears to be linked to ENaC activation through a Src kinase-dependent process.


Figure 11
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  		Fig. 11. Effect of an inhibitor of the Src family of tyrosine kinases, PP2, on ENaC activity and on the dopamine-induced increase in ENaC activity. In A, after control ENaC activity was recorded, 1 µM PP2 was applied to the apical and basolateral side of an L2 monolayer, and recording continued for ~5 min. Po was significantly increased by exposure to PP2 alone. However, when 10 µM dopamine was subsequently added to the apical side of the L2 monolayer, Po was not significantly increased above the level of PP2 treatment alone. Src activation requires 2 coordinate events: dephosphorylation of tyrosine 529 and phosphorylation of tyrosine 418. In B, we used specific antibodies to identify these phosphorylated forms of Src so that we could determine the relative amounts of inactive (phospho-Y-529), active (phospho-Y418), and total Src. Exposure of cells to dopamine reduces the amount of inactive Src and increases the amount of activated Src, even though the total amount of Src does not change substantially.

 
The inhibitors, PP1 or PP2, inhibit all members of the Src family of kinases. In epithelial cells, these could include Src, Yes, Yrk, Fyn, and possibly Lyn (85). Therefore, we wished to determine whether dopamine could activate Src, itself. Src activation requires two coordinate events: dephosphorylation of tyrosine 529 and phosphorylation of tyrosine 418. Specific antibodies can identify these phosphorylated forms of Src so that we could determine the relative amounts of inactive (phospho-Y-529), active (phospho-Y418), and total Src. Figure 11B shows that exposure of cells to dopamine reduces the amount of inactive Src, increases the amount of activated Src, even though the total amount of Src does not change substantially.

Dopamine increases ENaC activity through a mitogen-activated protein kinase (extracellular signal-regulated kinase)-dependent process. Src inhibitors can inhibit the Rap1-dependent activation of mitogen-activated protein kinases (MAPKs), or extracellular signal-regulated kinases (ERKs) (60), and dopamine can produce some of its effects through activation of ERK (11). In fact, dopamine activation of Na-K-ATPase is via a MAPKK/ERK-dependent process (27). Therefore, we examined the role of ERK by pretreating cells with the ERK inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126, 0.5 µM) for ~2 h. U0126 specifically inhibits MAPKK1 and MAPKK2 (MEK1 and MEK2) and, thus inhibits activation of ERK1 and ERK2 kinases. After pretreatment with U0126, channel activity was recorded before and after the treatment with 10 µM dopamine applied either to the apical side or basolateral side of the L2 monolayer. Unlike the dopamine-induced increase in Po in the absence of inhibitor, after U0126, Po was not increased by dopamine when applied to either surface (Fig. 12A).


Figure 12
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  		Fig. 12. Extracellularly regulated kinase (or ERK) is involved in the signaling pathway by which dopamine activates ENaC in L2 cells. L2 cells were pretreated with 0.5 µM U0126, an ERK inhibitor, for ~2 h. Then, the channel activity was recorded before and after the treatment with 10 µM dopamine applied to the apical side (A) of the L2 monolayer. Po was not increased by dopamine. B: treatment with dopamine activates ERK. Dopamine-treated and untreated cells were lysed, and Western blots probed for ERK or phospho-ERK. Whereas the total amount of ERK1/2 (the 2 bands are ERK 1 and ERK2) did not change, the amount of phosphorylated ERK increased in response to dopamine. C: neither D1 or D2 antagonists by themselves block ERK phosphorylation, but both together do. As in B, Western blots were probed for phospho-ERK in lysates from untreated cells, cells treated with 10 µM dopamine, 10 µM dopamine plus the D1 receptor blocker SCH-23390 (10 µM), 10 µM dopamine plus the D2 receptor blocker sulpiride (10 µM), or 10 µM dopamine plus both antagonists. Only both antagonists together significantly reduced ERK phosphorylation.

 
If dopamine-induced activation of Rap1 leads to activation of ERK1/2, then dopamine should induce an increase in ERK phosphorylation. We used ERK and phospho-ERK-specific antibodies to examine the effect of dopamine. Figure 12B shows that there is a large increase in phospho-ERK in response to dopamine despite there being little or no change in the total amount of ERK protein. When we examined the effect of receptor-specific dopamine antagonists, we found that neither the D1 antagonist SCH-23390 nor the D2 antagonist sulpiride completely blocked ERK phosphorylation, but a combination of both did (Fig. 12C). This suggests that both receptors are capable of promoting ERK phosphorylation.

The phosphatidylinositol 3-kinase inhibitor, LY-294002, also blocks the effect of dopamine. Phosphatidylinositol 3-kinases (PI3Ks) are ubiquitous lipid kinases that catalyze the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3-phosphate (86). All three of these molecules have been shown to activate ENaC (47, 92). Rap1 and ERK are both known to activate PI3K (15, 93). Therefore, we examined the role of PI3K by pretreating cells with the PI3K inhibitor LY-294002 (1 µM) for ~30 min. LY-294002 specifically inhibits PI3K and prevents the formation of all of the phosphatidylinositol-3-phosphates. After pretreatment with LY-294002, channel activity was recorded before and after the treatment with 10 µM dopamine applied to the apical side of the L2 monolayer. After LY-294002 treatment, Po was not increased by dopamine as it was in untreated cells (Fig. 13).


Figure 13
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  		Fig. 13. An inhibitor of phosphatidylinositol 3-kinase (PI3K) blocks the effect of dopamine. We examined the role of PI3K by pretreating cells with the PI3K inhibitor LY-294002 (1 µM) for ~30 min. LY-294002 specifically inhibits PI3K and prevents the formation of 3,4,5-phosphatidylinositol trisphosphate (PIP3). After pretreatment with LY-294002, channel activity was recorded before and after the treatment with 10 µM dopamine applied to the apical side of the L2 monolayer. After LY-294002 treatment, Po was not increased by dopamine as it was in untreated cells.

 
Dopamine promotes phosphorylation of dopamine response protein phosphatase 32 and dopamine-induced increases in ENaC activity requires phosphatase activity. The cAMP-EPAC-Rap1 pathway also activates the permissive, scaffolding protein 32-kDa dopamine response protein phosphatase (DARPP32) in epithelial cells other than lung (3, 4, 53). If DARPP32 were also playing a permissive role localizing the other components of the dopamine signaling pathway, then DARPP32 should be phosphorylated in response to dopamine. Figure 14A shows that dopamine does induce DARPP32 phosphorylation. Moreover, DARPP32 phosphorylation should be reduced by the activity of phosphatase 1 or 2A (16, 42, 58, 59). Interestingly, phosphatases have also been implicated in dopamine activation of Na-K-ATPase so we examined the involvement of protein phosphatases in dopamine activation of ENaC. We applied an inhibitor, okadaic acid, that blocks the most common phosphatases, PP1 and PP2A. Like Src kinase, there appears to be a constitutive inhibition of ENaC by one of the phosphatases since inhibiting phosphatases with okadaic acid alone produces a significant increase in ENaC activity (Fig. 14B). However, after addition of okadaic acid, dopamine was ineffective at increasing ENaC activity (Fig. 14B). Thus dopamine D1 receptor activation appears to be linked to ENaC activation through DARPP32, and DARPP32 activation is a phosphatase-dependent process.


Figure 14
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  		Fig. 14. The 32-kDa dopamine receptor protein phosphatase DARPP32 is activated by dopamine, and the DARPP32 and phosphatase 1 and 2A inhibitor okadaic acid blocks the dopamine-induced increase in ENaC activity. A: treatment with dopamine activates DARPP32 (arrow). Dopamine-treated and untreated cells were lysed, and Western blots probed for DARPP32 or phospho-DARPP32. Although the total amount of DARPP32 did not change, the amount of phosphorylated DARPP32 increased in response to dopamine. In B, after control ENaC activity was obtained, 10 µM okadaic acid was applied to the apical and basolateral side of a L2 monolayer and recording continued for ~5 min. Po was significantly increased by exposure to okadaic acid alone. However, when 10 µM dopamine was subsequently added to the apical side of the L2 monolayer, Po was not significantly increased above the level of okadaic acid treatment alone.

 

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L2 cells are a good model for AT2 cells. Although it is possible to isolate and grow AT2 cells from rat and mouse lungs in primary culture, there are many occasions when having a continuous cell line that has AT2 cell characteristics could be very useful (29, 39, 84). To this end, a variety of different cell types have been used, all have positive and negative features: A549 are a human alveolar lung carcinoma line, but they no longer are contact inhibited and do not form tight junctions; H441 are of human origin, form monolayers, and transport Na, but are derived from Clara cells rather than alveolar type 1 or 2 cells; 16HBE14 are human bronchial epithelial cells; and Calu-3 cells are human cells but derived from serous airway epithelial cells. In contrast, L2 cells are derived from AT2 cells that have spontaneously transformed and, therefore, have maintained their contact inhibition and ability to produce surfactant protein. As we have shown in this paper, they also have transport and single channel characteristics indistinguishable from primary cultures of AT2 cells. More to the point, they have levels of dopamine receptor proteins, D1 and D2, comparable to AT2 cells (Figs. 5 and 6). However, they do not form monolayers with as high a resistance as native AT2 cells in primary culture. Nonetheless, as we have demonstrated, it is possible to measure amiloride-sensitive transepithelial current comparable to that measured in AT2 monolayers.

Coordinate regulation of apical and basolateral Na transport in lung epithelium. Lung epithelium is different from most other Na-transporting epithelia in the sense that the lung is not involved in net salt reabsorption, but rather, the lung is responsible for maintaining an exquisite balance of reabsorption and secretion that maintains a closely controlled level of alveolar (and airway) fluid (9, 10, 61). Under these circumstances, having control of both the Na channels associated with the entry step as well as the Na-K-ATPase associated with the exit step might be useful under different physiological circumstances (61, 83). In contrast, strongly increasing the activity of the basolateral Na-K-ATPase alone would not produce an increase in Na transport comparable to the increase in pump activity if the entry step becomes rate limiting for Na transport. In the final analysis, maximal Na transport will occur under circumstances when the activity of the Na-K-ATPase increases approximately in parallel with the activity of apical Na channels. Therefore, after the fact, maybe it is not so surprising that dopamine increases the activity of both apical ENaC and basolateral Na-K-ATPase, but it does seem surprising that the activation of the two pathways seem to share so many common mechanisms. In this sense, Guerrero et al. (27) reported that stimulation of D1 dopamine receptors on the basolateral surface of AT2 cells resulted in activation of Na-K-ATPase via MAPK/ERK. This effect was mediated by Ras proteins, the serine/threonine kinase Raf-1, and diacylglycerol-dependent PKC isoenzymes, but this pathway did not involve the Grb2-Sos complex formation (28). In addition, as we have shown for dopamine activation of ENaC, they have demonstrated that dopamine activation of Na-K-ATPase is dependent upon a protein phosphatase (40). We have now shown in this paper that dopamine stimulation of ENaC through D1 receptors is inhibited by ERK and PI3K inhibitors. This work demonstrates an effect of dopamine on Na channels that is consistent with a coordinated response of Na-K-ATPase and Na channels to promote maximal increases in transport and alveolar fluid clearance.

Localization of the components of the dopamine receptor signaling pathway. Apical dopamine binds to and activates apical D1 receptors. D1 receptors couple to Gs proteins to activate adenylyl cyclase and increase intracellular cAMP (Fig. 9), but we have previously shown that activation of basolateral beta2-adrenergic receptors also activates Gs and increases cAMP (12). However, the result of increasing cAMP in response to these two different classes of agonists is profoundly different: apical dopamine increases ENaC Po, but beta2-adrenergic agonists cause a significant increase in the number of channels (12). The implication is that receptor-induced cAMP is somehow or other capable of selectively activating one pathway, but not the other. In this context, there is evidence for substantial compartmentalization of the two cAMP signaling pathways, EPAC and PKA (8, 13, 37, 52, 66). Indeed, fluorescently labeled EPAC and PKA constructs have been used to demonstrate this localization (66). The implication for our work is that apical dopamine produces cAMP in a compartment that is available to activate EPAC but not PKA, whereas beta2-adrenergic agonists generate cAMP in a compartment where it can activate PKA but not EPAC. Figure 9 appears to reflect this compartmentalization. Forskolin induces a very large increase in cAMP presumably because it is activating all adenylyl cyclase in the cells. Dopamine, on the other hand, produces an significant, but smaller, increase in cAMP because it activates adenylyl cyclase in only a restricted apical compartment. A schematic diagram of the entire dopamine pathway beginning with D1 receptor activation is shown in Fig. 15.


Figure 15
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  		Fig. 15. Schematic diagram of dopamine signaling pathways. Initially dopamine binds to a D1 receptor that couples to and activates Gs protein. The activated G protein via the {alpha}-subunit activates adenylyl cyclase to produce cAMP. cAMP activates EPAC. At the same time, the G protein (probably via its beta{gamma}-subunits) activates an Src family kinase. Both EPAC and Src activate the small G protein, Rap1. Rap1 initiates 2 separate pathways. The first is to activate PI3K that produces PIP3, which activates ENaC. The second is activation of ERK which independently alters PI3K activity. Increased PI3K activity produces more PIP3, a known activator of ENaC. All of these events depend upon the proper localization of the signaling elements that is promoted by the cAMP-activated scaffolding protein DARPP32. DARPP32 activity is regulated by the activity of other protein phosphatases that can be blocked by okadaic acid.

 
Constitutive activity of Src and Src dependence of dopamine activation of ENaC. Figure 11 shows that the Src inhibitor PP2 blocks the action of dopamine implying that dopamine activation of Gs protein in L2 cells activates Src, which is at least partially responsible for ENaC activation. Src could be activated by two alternative mechanisms. Activation of Gs (and other G proteins) by D1 receptors releases the {alpha}-subunit of the G protein, but also the beta{gamma}-subunits. Interestingly, G protein beta{gamma}-subunits are capable of activating Src kinases (5, 32, 43, 56, 88). The second possibility is that an increase in cAMP itself can activate Src (44, 60, 72, 81). For either pathway the implication is that there are redundant mechanisms, EPAC and Src, both dependent upon D1 receptor activation for activation of Rap1. We would expect that the EPAC activation of Rap1 to be fast and the Gbeta{gamma} activation to be slow and sustained; therefore, together the two pathways could produce a rapid and sustained response to dopamine.

However, Fig. 11 also shows that the Src inhibitor by itself also significantly increases the Po of ENaC. This implies that there is constitutive Src kinase activity that reduces ENaC activity but also that there must be two separate functions for Src-like kinases: a constitutive activity that reduces ENaC activity and dopamine activation that increases activity. These two separate functions may not represent a contradictory role for Src itself. The inhibitor PP2 inhibits all members of the Src kinase family, of which there are at least four members present in epithelial cells (Src1, Src2, Fyn, and Yes) (85). There has been at least one report that suggests that the tonic inhibitory role might be due to the Yes kinase (23, 55). Because the same is likely to be true in L2 cells, the stimulatory role of dopamine must be due to one of the other members of the family (Fyn or Src1/2). Our experiments suggest that Src1/2 are activated in response to dopamine (Fig. 11B), but it is still possible that one of the other Src family members also contributes to the dopamine response. Only additional experiments that target the individual kinases with approaches such as an antisense or siRNA similar to that used by Yue et al. (91) will definitively resolve the relevant kinase.

ERK and ENaC activity. Rap1 activation by EPAC or Src activates ERK1/2 (Fig. 12). This is a well-described pathway (20, 24, 37, 81, 90). Once it was clear that dopamine activation of ENaC involved a member of the Src family of tyrosine kinases, it was not too surprising that MAPKs were also involved since there have been numerous reports that describe Src activation of MAPKs (62) (whether or not the activation is Rap1 mediated). Activation of the ERK cascade is particularly interesting since ERK does phosphorylate ENaC (74). However, this pathway seems more likely to be associated with Src-mediated constitutive inhibition of ENaC since ERK phosphorylation of ENaC reportedly inhibits ENaC function and, in fact, inhibits function by changing the number of ENaC at the surface membrane rather than by changing the Po of the channel (30, 74, 79). Therefore, it seems likely that the MAPK-mediated increase in Po by dopamine must involve one of the other MAPK cascades, either p38 or MEK kinases.

PI3K is necessary for the dopamine-induced increase in ENaC activity. We also found that dopamine activation of ENaC could be blocked by inhibitors of PI3K. PI3K in epithelial tissue is usually activated by a Ras protein. In particular, aldosterone activation of ENaC involves activation of K-Ras that subsequently activates PI3K (2, 49, 78), and K-Ras activation can activate ENaC (80). In addition, Guerrero et al. (28) have shown that D1 receptor activation leads to Ras activation that does not depend upon the signaling intermediates Grb2 and Sos. Moreover, Src kinases are known to activate Ras proteins in a Grb2-Sos-independent manner (21, 38, 45, 65). Thus, once Src is activated, it is not surprising that PI3K might also be activated. However, while Ras may be involved in PI3K activation, there is substantial evidence that activation of Rap1 alone is sufficient to activate PI3K (86, 89). We have previously shown that PI3K-mediated production of PIP3 in other epithelial cells is necessary to sustain ENaC activity (47) and application of PIP3 to the cytosolic surface of apical membrane patches greatly increases ENaC activity (92). Thus, one mechanism by which dopamine increases ENaC activity is by increasing the amount of membrane PIP3. Besides increasing ENaC activity, PIP3 is important for promoting exocytosis and regulated insertion of proteins in cell membranes. Because dopamine increases Na-K-ATPase activity by promoting insertion of new pump units (67), PIP3 could be the common agent that increases ENaC activity and Na-K-ATPase activity in parallel.

In summary, dopamine, either endogenous or clinically applied, has a potential to increase Na reabsorption by increasing both the Po of apical ENaC in the apical membranes and the activity of Na-K-ATPase in the basolateral membrane of alveolar epithelial cells. This increase appears to be a coordinated response of the alveolar epithelium to maximize the amount of Na transport and subsequent water reabsorption.


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This work was supported by National Institutes of Health Grants R24DK-064399 to D. C. Eaton, T32AA-013528 support to M. N. Helms, and R01HL-071621, R01HL-063306, and P50AA-013757 to D. C. Eaton and L. Jain.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. C. Eaton, Dept. of Physiology, Emory Univ. School of Medicine, 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.


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