Dopamine regulates Na-K-ATPase in alveolar epithelial cells via MAPK-ERK-dependent mechanisms

Carmen Guerrero1,2, Emilia Lecuona1, Liuska Pesce1, Karen M. Ridge1, and Jacob I. Sznajder1

1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, Illinois 60611; and 2 Centro de Investigación del Cáncer, Universidad de Salamanca, 37007 Salamanca, Spain


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dopamine (DA) increases lung edema clearance by regulating vectorial Na+ transport and Na-K-ATPase in the pulmonary epithelium. We studied the role of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway in the DA regulation of Na-K-ATPase in alveolar epithelial cells (AEC). Incubation of AEC with DA resulted in a rapid stimulation of ERK activity via dopaminergic type 2 receptors. Analysis of total RNA and protein showed a 1.5-fold increase in the Na-K-ATPase beta 1-subunit mRNA levels and up to a fivefold increase in beta 1-subunit protein abundance after DA stimulation, which was blocked by the MAPK kinase (MEK) inhibitors PD-98059 and U-0126. Also, the DA-ERK pathway stimulated the synthesis of a green fluorescent protein reporter gene driven by the beta 1-subunit promoter, which indicates that DA regulates the Na-K-ATPase beta 1-subunit at the transcriptional level. The DA-mediated increase in beta 1-subunit mRNA protein resulted in an increase in functional Na pumps in the basolateral membranes of alveolar type II cells. These results suggest that the MAPK-ERK pathway is an important mechanism in the regulation of Na-K-ATPase by DA in the alveolar epithelium.

alveolar type II cells; green fluorescent protein; transcriptional regulation; basolateral membrane


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NA-K-ATPASE IS A UBIQUITOUS enzyme essential for the maintenance of membrane potential and control of cellular volume (17). It also has been shown to have an important role in lung edema clearance by increasing active Na+ transport (4, 23). A functional Na-K- ATPase is a transmembrane heterodimer protein composed of two subunits (alpha  and beta ) (29). The catalytic alpha -subunit contains the binding sites for Na, K, and ATP. The glycosylated beta -subunit appears to be important in the insertion of the Na pump to the plasma membrane (20). In alveolar type II (ATII) cells, Na-K-ATPase is predominantly composed of alpha 1- and beta 1-isoforms (25).

Na-K-ATPase can be regulated by short-term or long-term mechanisms. The short-term regulation of the Na pump involves changes in its turnover, its affinity for substrates, and/or its abundance at the cell surface (7, 8, 12). The long-term regulation of Na-K-ATPase includes transcriptional activation, translation, and protein stability (12). Aldosterone, insulin, thyroid hormone, epidermal growth factor, and keratinocyte growth factor are some of the agonists known to regulate the transcriptional and/or translational rate of the Na pump, although the molecular mechanisms involved have not yet been elucidated.

Dopamine (DA) inhibits Na-K-ATPase activity in most tissues, including brain (6), vascular bed (24), and kidney (5). In contrast, recent studies have suggested that DA increases the ability of the lungs to clear edema, probably by the regulation of the alveolar epithelial Na-K-ATPase function (1, 27).

The mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. ERK proteins (ERK1/2) have many substrates, including the ternary complex factor Elk-1, a member of the Ets family of transcription factors, which is recruited by serum-response factors to bind serum-response elements, located in the promoters of many early genes (31) and also in the 5'-flanking region of the Na-K-ATPase beta 1-subunit gene (18).

DA has an inhibitory role on the ERK pathway in most cell types (22, 30), although recent reports suggested that DA activates ERK proteins in Chinese hamster ovary cells (32) and neurons (14). However, there are no studies exploring whether the activation of ERK by DA regulates Na-K-ATPase.

We have investigated whether DA activates the ERK signaling pathway in alveolar epithelial cells (AEC) and whether this activation regulates Na-K-ATPase. Our results demonstrate that DA regulates beta 1-subunit mRNA and protein abundance via an ERK-dependent pathway that involves dopaminergic type 2 receptors (D2R) but not D1R and that this stimulation results in an increase in protein abundance in the basolateral membranes (BLMs) of ATII cells where the Na pump is active and, consequently, in an increase in Na-K-ATPase activity.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation and culture. ATII cells were isolated from pathogen-free male Sprague-Dawley rats as previously described (23, 25). The day of isolation and plating was designated culture day 0. All experimental conditions were tested in day 2 cells.

ERK assay. ATII cells were serum starved for 18-24 h and treated with regulators for the desired time. The ERK activity was determined as phosphorylation of the ERK substrate Elk-1 using a p44/p42 MAPK assay kit (New England Biolabs, Beverly, MA). PD-98059 (New England Biolabs) and U-0126 (Promega, Madison, WI) were used as inhibitors of the ERK activity.

Cloning of the Na-K-ATPase beta 1-subunit promoter into pEGFP-1 plasmid. A 786-bp fragment of the Na-K-ATPase beta 1-subunit gene promoter region containing 773 bp upstream and 12 bp downstream from the nucleotide +1 was subcloned into the EcoRV site of plasmid pT7blue(R) (Novagen, Madison, WI) and then subcloned as a HindIII-EcoRI fragment into the promoter reporter vector pEGFP-1 (Clontech, Palo Alto, CA). The final construct was named pEGFP-beta 1P.

Generation of stable cell lines overexpressing pEGFP-beta 1P construct. A549 cells derived from human AEC (American Type Culture Collection CCL 185) were transfected with 2-5 µg of plasmid pEGFP-beta 1P by using SuperFect Reagent (QIAGEN, Valencia, CA) and selected in the presence of 600 µg/ml Geneticin (G418).

RNA isolation and analysis by semiquantitative RT-PCR. Total cellular RNA from ATII cells was isolated by using RNeasy total RNA kit (QIAGEN). The reverse transcription (RT) reaction was performed using the SUPERSCRIPT preamplification system (GIBCO BRL, Grand Island, NY). The resultant cDNAs were amplified by PCR using specific primers and analyzed by agarose gel electrophoresis. The amplified bands were quantified by densitometric scan and normalized against the internal control gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Oligonucleotides and conditions used in PCR. For the amplification of the Na-K-ATPase beta 1-subunit, we used the following set of oligonucleotides: 5'-AAT CAT GAA CGA GGA GAG CG-3' and 5'-AGG TGA GGT TGG TGA ACT GC-3' that correspond to the positions 418-437 and 786-805, respectively, of the cDNA from the rat gene (considering as number 1 the "A" of the first ATG of the gene). The conditions for the PCR were 94°C, 1 min; 53°C, 1 min 30 s; and 72°C, 2 min for 41 cycles. For the amplification of the control gene GAPDH, we used the rat GAPDH Control Amplimer Set (Clontech) at 94°C, 45 s; 60°C, 45 s; and 72°C, 2 min for 21 cycles. Primers used for the detection of D2R were 5'-CCT TCA CCA TCT CTT GC-3' and 5'-CCT TCT GCT GGG AGA GC-3', corresponding to positions 488-504 and 1089-1105, respectively, of the cDNA from the mouse gene, and the conditions for the PCR were 94°C, 1 min; 53°C, 1 min 30 s; and 72°C, 2 min for 30 cycles.

Quantitation of green fluorescent protein fluorescence. pEGFP-1- or pEGFP-beta 1P-expressing A549 cultures were plated in four-well Permanox chamber slides (Nalge Nunc International, Naperville, IL), starved, and treated with DA or DA plus PD-98059 for 24 h. Cells were fixed by incubation with 4% formaldehyde in PBS for 15 min, washed with PBS, and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA).

Photographs were taken using a GFP (green fluorescent protein) filter set in a Nikon Eclipse E800 fluorescence microscope equipped with a 100-W mercury lamp. DA effect was evaluated by visually comparing the regional level of fluorescence in pictures of cultures. Quantitative data were obtained by fluorometric assay and immunoblotting analysis.

A549 clones expressing pEGFP-beta 1P or transfected with control plasmid pEGFP-1 were plated in 6-cm plates, starved at subconfluence, and treated with DA or DA plus PD-98059 for 24 h. Cell cultures were washed twice in PBS and resuspended in 300 µl of sonication buffer (50 mM NaH2PO4, 10 mM Tris · HCl, and 200 mM NaCl), pH 8.0. Twofold serial dilutions ranging from 50 to 3.125 µg were prepared in sonication buffer. Fluorescence intensity was measured in a Perkin-Elmer fluorescence spectrometer (model LS-3B; Perkin-Elmer, Oakbrook, IL) using an excitation filter of 490 nm and an emission filter of 510 nm.

Deglycosylation of the Na-K-ATPase beta 1-subunit protein. Total protein (100 µg) from ATII cells was digested with 1 U of N-glycosidase F as described previously (15).

Western blot analysis. Total deglycosylated protein (100 µg) or 5 µg of BLMs, isolated as described previously (8), were resolved by 12.5% SDS-PAGE and analyzed by immunoblotting using specific Na-K-ATPase anti-beta 1 polyclonal antibody or anti-alpha 1 monoclonal antibody (a generous gift from Dr. Martin-Vasallo, University of La Laguna, Spain, and Dr. M. Caplan, Yale University, CT, respectively). For the detection of GFP, 25 µg of total protein were resolved by 12.5% PAGE and analyzed by immunoblotting using an anti-GFP monoclonal antibody, clone B34 (BABCO, Richmond, CA).

Transport measurements. Ouabain-sensitive 86Rb+ uptake was used to estimate the rate of K+ transport by Na-K-ATPase in AEC. Briefly, cells were preincubated for 5 min at 37°C in a gyratory bath at 100 rpm by adding 5 mM ouabain in HEPES-buffered DMEM. This medium was then removed, and otherwise identical fresh medium containing 1 µCi/ml 86Rb+ was added. After a 5-min incubation (37°C, 100 rpm), uptake was terminated by aspirating the assay medium and washing the plates in ice-cold MgCl2. Plates were allowed to dry, and cells were solubilized in 0.2% SDS. 86Rb+ influx was quantified in aliquots of the SDS extract with a liquid scintillation counter. Protein was quantified in aliquots by the Lowry method.

Statistical analysis. Data are means ± SE. All statistical analyses were made using one-way ANOVA, except data in Figs. 4 and 6 that were treated with two-way ANOVA (time and DA as the 2 independent variables), followed by a multiple comparison test (Tukey) when the F statistic indicated significance. Results were considered significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DA activates MAPK-ERK in ATII cells. Figure 1 shows that DA induced up to 3.5-fold increase in ERK activity after 5 min of incubation of subconfluent, serum-starved ATII cells. This activation was specific because it was abolished when cells were pretreated with the specific MAPK kinase (MEK) inhibitor PD-98059 2 h before DA stimulation.


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Fig. 1.   Dopamine (DA)-stimulated extracellular signal-regulated kinase (ERK) activity in alveolar type II (ATII) cells. Serum-starved ATII cells were incubated with 10 µM DA in the presence and absence of the mitogen-activated protein kinase kinase (MEK) inhibitor PD-98059 (50 µM), and the ERK activity was determined as phosphorylation of the transcription factor Elk-1 (relative values). Top: means ± SE; n = 4; **P < 0.01 vs. control (CT). Bottom: representative ERK assay. P-Elk-1, phosphorylated Elk-1.

DA stimulates ERK activity via D2R. RT-PCR analysis of total RNA showed that D1R (2) and D2R mRNA are both expressed in ATII cells (Fig. 2). To determine whether D1R or D2R receptors mediate ERK activation by DA, serum-starved ATII cells were treated with DA in the presence of specific D1R and D2R antagonists (Sch-23390 and S-sulpiride, respectively) or with a D1R agonist (fenoldopam) and D2R agonists [quinpirole and R(-)-propylnorapomorphine hydrochloride (R-NPA)]. As shown in Fig. 3, preincubation with S-sulpiride (but not with Sch-23390) inhibited the activation of ERK by DA. Also, D2R activation by quinpirole or R-NPA, but not by the D1R agonist fenoldopam, stimulated ERK activity in ATII cells.


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Fig. 2.   ATII cells expressed dopaminergic type 2 receptor (D2R). Size analysis of PCR products in 2% agarose gel stained with ethidium bromide is shown. Amplification product of the predicted size (600 bp) for D2R was evident in the RT-PCR. Madin-Darby canine kidney (MDCK) cells were used as a positive control of D2R expression. 1, A549; 2, ATII; 3, MDCK; M, molecular weight marker, 1-kb DNA ladder.



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Fig. 3.   DA-stimulated ERK activity via D2R but not D1R. Serum-starved ATII cells were treated with 10 µM DA for 5 min in the presence and absence of the D1R antagonist Sch-23390 or the D2R antagonist S-sulpiride (both added at 10 µM 30 min before DA stimulation), and the ERK activity was determined. Cells also were treated with D1R agonist fenoldopam (10 µM, 5 min) and D2R agonists quinpirole and R(-)-propylnorapomorphine hydrochloride (R-NPA, 10 µM, 5 min). Top: means ± SE; n = 3; **P < 0.01 vs. CT. Bottom: representative ERK assay.

beta -Adrenergic receptors (beta -ARs) activate ERK proteins via a G protein-dependent mechanism (11). DA has been found to activate beta -ARs at high doses (9), but pretreatment of ATII or A549 cells with 10 µM propanolol, a specific beta -AR antagonist, did not affect the DA-mediated ERK stimulation (data not shown), which indicates that the observed DA effect is not mediated by the beta -adrenergic pathway.

DA regulates Na-K-ATPase beta 1-subunit mRNA and protein abundance in ATII cells via an ERK-dependent mechanism. To determine whether DA regulates Na-K-ATPase at the mRNA and/or protein level and whether this effect is mediated by ERK activation, serum-starved ATII cells were incubated with DA in the presence and absence of the MEK inhibitor PD-98059. Changes in mRNA and protein levels were examined at different times. As shown in Fig. 4, there was a 1.5-fold increase in the Na-K-ATPase beta 1-subunit mRNA levels after ~18 h of DA stimulation. This increase was abolished in cells preincubated with PD-98059. The optimal conditions for the PCR of both the Na-K-ATPase beta 1-subunit gene (41 cycles) and the GAPDH gene (21 cycles) were established in preliminary studies (Fig. 5).


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Fig. 4.   DA increased the Na-K-ATPase beta 1-subunit mRNA via ERK proteins. Serum-starved ATII cells were incubated with 10 µM DA in the presence and absence of the MEK inhibitor PD-98059 (50 µM; I) for the indicated times. mRNA from stimulated cells was analyzed by semiquantitative RT-PCR using specific beta 1 primers. Amplification of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as internal control. Top: means ± SE; n = 3; **P < 0.05 vs. control. Bottom: representative PCR amplification of beta 1 and GAPDH genes.



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Fig. 5.   Linear range of Na-K-ATPase beta 1-subunit amplification by PCR. Same amount of an RT reaction from ATII cells of total RNA was used to amplify the Na-K-ATPase beta 1-subunit gene and the GAPDH gene in a PCR. Top: aliquots from the reaction were taken at different cycles and fractionated by electrophoresis in a 1.5% agarose gel. Bottom: log of the DNA band density was plotted as a function of the number of cycles.

Time-course studies demonstrated that DA increases Na-K-ATPase beta 1-subunit protein abundance in whole cell homogenates with a maximum (~5-fold) effect at 24 h (Fig. 6), which correlates with the maximum increase in mRNA abundance observed at 18 h (Fig. 4). The DA-mediated increased in beta 1-subunit protein abundance was dependent on ERK proteins because it was inhibited by PD-98059 (Fig. 6) and also by the newly described MEK inhibitor U-0126 (Fig. 7A). Additionally and according to results in Fig. 3, treatment with quinpirole for 24 h also increased total beta 1-subunit protein abundance in an ERK-dependent manner (Fig. 7B). No significant changes were observed in the Na-K-ATPase alpha 1-subunit mRNA or protein levels in whole cell homogenates (data not shown).


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Fig. 6.   DA increased Na-K-ATPase beta 1-subunit protein abundance via an ERK-dependent mechanism. ATII cells were serum starved and treated with 10 µM DA for the indicated times in the presence and absence of the MEK inhibitor PD-98059 (I). Western blot of whole cell homogenates of total deglycosylated protein was performed using anti-beta 1 polyclonal antibody. Top: means ± SE; n = 3; *P < 0.05, **P < 0.01 vs. CT. Bottom: representative Western blot.



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Fig. 7.   U-0126 inhibits DA- and quinpirole (QP)-mediated Na-K-ATPase beta 1-subunit expression. ATII cells were starved and treated with 10 µM DA or quinpirole for 24 h in the presence and absence of 10 µM of the MEK inhibitor U-0126. A and B: representative Western blots showing beta 1-subunit protein expression in whole cell homogenates. C: representative Western blot showing green fluorescent protein (GFP) expression.

DA regulates the transcription of the Na-K-ATPase beta 1-subunit. To determine whether DA regulates the Na-K-ATPase beta 1-subunit at the transcriptional level, permanent transfectants of A549 cells expressing pEGFP-beta 1P or vector alone were serum starved and treated with DA in the presence and absence of PD-98059 or U-0126. DA activated ERK in A549 cells to the same extent as in ATII cells (Fig. 8A). Cells were analyzed by 1) fluorescent microscopy, 2) fluorescent spectrophotometry, and 3) Western blot with anti-GFP antibodies. The three different approaches showed that DA increased GFP expression under the control of the Na-K-ATPase beta 1-subunit promoter (Fig. 8B). The increase observed in both the fluorometric analysis (Fig. 8B, b) and the immunoblotting (Fig. 8B, c) was ~1.5-fold, which agrees with the increase in mRNA detected by PCR (Fig. 4). The DA-induced increase in GFP expression was inhibited by PD-98059, as demonstrated by Western blot (Fig. 8B, c). We found that PD-98059 is autofluorescent, precluding the ability to evaluate quantitatively its inhibitory effect on DA-induced GFP expression by fluorometric analysis. Additionally, in accordance with results in Fig. 3, quinpirole also induced GFP expression, which was inhibited by U-0126 (Fig. 7C). Taken together, these results demonstrate that DA regulates Na-K-ATPase beta 1-subunit at the transcriptional level by an ERK-dependent mechanism.


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Fig. 8.   A: DA stimulates ERK activity in A549 cells to the same extent as in ATII cells. Subconfluent ATII and A549 cells were serum starved for 20-24 h and treated with 10 µM DA for 5 min, and the ERK activity was assayed in the lysates. Numbers beneath each band indicate the relative level of activation with respect to control. B: DA stimulates the transcription of GFP under the control of the beta 1-promoter. a, images of A549 cells transfected with promoterless plasmid pEGFP-1 treated with DA for 24 h (1); pEGFP-beta 1P untreated (2) or treated with DA for 24 h (3). b: relative fluorescence intensity of twofold serial dilution of suspended A549 cells expressing pEGFP-beta 1P or pEGFP-1 plasmids untreated or treated with DA. RFU, relative fluorescence units. Each line represents the means ± SE of 4 independent experiments. ***P < 0.001 vs. CT pEGFP-beta 1P. dagger dagger dagger P < 0.001 vs. CT pEGFP-1. The relative fluorescence units were plotted as a function of the log of total protein (in µg). c: Western blot analysis of A549 clones expressing pEGFP-beta 1P treated with DA or with DA plus PD-98059. Top: means ± SE; n = 6; *P < 0.05 vs CT. Bottom: representative Western blot showing GFP.

DA increases Na-K-ATPase protein abundance and activity in BLMs. BLMs were isolated from serum-starved ATII cells treated with DA for 24 h in the presence or absence of PD-98059. Immunoblotting with anti-alpha 1 and anti-beta 1 antibodies showed an increase in both Na-K-ATPase subunits in BLMs of cells treated with DA compared with control cells and cells pretreated with PD-98059 (Fig. 9A). This increase in Na pump abundance at BLMs resulted in an increase in functional pumps; as shown in Fig. 9B, Na-K-ATPase activity (measured as 86Rb+ uptake) increased in serum-starved ATII cells 24 h after treatment with DA, an effect prevented by PD-98059.


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Fig. 9.   DA increases Na-K-ATPase activity and Na pump abundance in basolateral membranes (BLMs) of ATII cells. A: ATII cells were serum starved and treated with 10 µM DA for 24 h in the presence and absence of the MEK inhibitor PD-98059 (I). Western blot of BLMs was performed using anti-alpha 1 or anti-beta 1 antibodies. Top: means ± SE; n = 3; *P < 0.05, **P < 0.01 vs. CT. dagger P < 0.01 vs. DA. Bottom: representative Western blot. B: Na-K-ATPase activity increased in cells treated with DA, which was prevented by PD-98059; means ± SE; n = 3; *P < 0.05. **P < 0.05 vs. DA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are that DA regulates Na-K-ATPase beta 1-subunit via a long-term mechanism that results in a functional Na-K-ATPase dimer in the BLMs of AEC and that these events involve D2R but not D1R.

In animal models, the fluid is reabsorbed from the lungs predominantly by active Na+ transport via the apical Na+ channels and basolateral Na-K-ATPases located in the AEC. There is a large body of information regarding regulation of Na-K-ATPase in the kidney but much less is known about its regulation in the alveolar epithelium. Catecholamines, in particular beta -adrenergic agonists, have been shown to regulate Na+ channels and Na-K-ATPase activity in lung alveolar epithelium (8, 19). Recent studies have demonstrated that DA stimulates lung edema clearance, possibly via modulation of Na-K-ATPase in the pulmonary epithelium (1, 27).

DA-mediated regulation of Na-K-ATPase by short-term mechanisms has been reported in kidney (5, 7) and brain (6). We are reporting for the first time that DA stimulates ERK activity in ATII cells and that this activation results in the transcriptional regulation and translation of the Na-K-ATPase beta 1-subunit via a MAPK-ERK-dependent mechanism.

In most systems, DA inhibits Na-K-ATPase activity by short-term mechanisms via the dopaminergic receptors D1R and D2R (5, 6, 14). In the kidney, stimulation of D1R inhibits Na-K-ATPase via the cAMP-protein kinase A pathway (12), a negative regulator of ERK activation (10, 28). On the contrary, DA-mediated D1R activation appears to regulate Na-K-ATPase in AEC within 15 min by short-term regulatory mechanisms (26). However, as shown in Fig. 3, fenoldopam, a D1R agonist, did not alter basal ERK activation, and preincubation of ATII cells with the D1R antagonist Sch-23390 did not affect the stimulation of ERK by DA. In contrast, inhibition of D2R by S-sulpiride, a specific D2R antagonist, resulted in inhibition of DA-stimulated ERK activity. In agreement with these results, stimulation of ATII cells with the D2R agonists quinpirole and R-NPA activated ERK to similar levels as DA. Activation of ERK proteins by D2R stimulation also has been recently described in neuronal tissue (33), but this is the first study showing that activation of ERK via D2R results in transcriptional regulation of Na-K-ATPase.

Our results showing that DA stimulation is associated with augmented Na-K-ATPase mRNA and protein abundance are similar to a recent report where beta -adrenergic agonists increased Na-K-ATPase alpha 1-subunits in AEC (21). However, that study did not identify the mechanisms regulating this event. In the present study, the Na-K-ATPase beta 1-subunit mRNA levels increased by 1.5-fold after ~18 h of incubation with DA (Fig. 4) without change in the alpha 1-subunit mRNA levels. This increase is due to transcriptional activation because it is similar to the increase observed in GFP synthesis under the control of the beta 1-subunit promoter (Figs. 7C and 8B). We also found that Na-K-ATPase beta 1-subunit protein increased by up to fivefold after incubation with DA (Fig. 6). The larger increase in protein abundance compared with the mRNA levels may suggest that DA probably has additional effects at posttranscriptional or translational levels. In this line, parallel studies indicate a role of a posttranscriptional rapamycin-sensitive mechanisms involved in the regulation of the Na-K-ATPase by beta -adrenergic agonists (Pesce L, unpublished data). The existence of posttranscriptional regulatory mechanisms has been reported previously in the regulation of the Na pump (12). Our results are consistent with previous studies suggesting that upregulation of the beta 1-subunit mRNA can occur without modification of the Na-K-ATPase alpha 1-subunit mRNA levels (3, 13, 16). Also, the fact that both alpha 1- and beta 1-subunits increase in the BLMs of ATII cells treated with DA, resulting in increased Na-K-ATPase activity (see Fig. 9), suggests that the DA-mediated regulation of the Na pump is physiologically relevant. We reason that the increase in alpha 1-subunit in ATII cell BLMs can be due to recruitment of proteins from intracellular pools as described previously (8).

In summary, these results demonstrate that in AEC, DA regulates Na-K-ATPase by ERK proteins via the D2R by a mechanism that involves de novo synthesis of beta -subunits and possibly recruitment of preexisting alpha -subunits. This is the first report that links ERK activation to Na-K-ATPase regulation. Further studies are needed to elucidate the posttranscriptional regulatory mechanisms involved in the DA-mediated Na-K-ATPase regulation.


    ACKNOWLEDGEMENTS

We thank Dr. Aparajita Ghosh for technical help and Dr. Eugenio Santos for valuable discussions.


    FOOTNOTES

This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-65161 and the American Lung Association of Metropolitan Chicago.

Address for reprint requests and other correspondence: J. I. Sznajder, Pulmonary and Critical Care Medicine, Northwestern Univ., 300 E. Superior St. Tarry 14-707, Chicago, IL 60611 (E-mail: j-sznajder{at}northwestern.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 April 2000; accepted in final form 14 February 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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