Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of alpha -subunits in lung alveolar cells

Alejandro M. Bertorello1, Karen M. Ridge2, Alexander V. Chibalin1, Adrian I. Katz3, and Jacob I. Sznajder2

1 Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden; 2 Department of Pulmonary and Critical Care Medicine, Michael Reese Hospital, University of Illinois at Chicago, Chicago 60616; and 3 Department of Medicine, University of Chicago, Chicago, Illinois 60637

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Catecholamines promote lung edema clearance via beta -adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to beta -adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 µM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 µM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase alpha -subunit; rather, it was the result of an increased number of alpha -subunits recruited from the late endosomes into the plasma membrane. The recruitment of alpha -subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting alpha -subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.

alveolar epithelium; protein kinases; actin cytoskeleton; sodium transport; early endosomes; late endosomes

    INTRODUCTION
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Abstract
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Results
Discussion
References

PULMONARY EDEMA FORMATION is due to increased hydrostatic pressure in the pulmonary circulation or increased pulmonary capillary permeability (43). Regardless of its pathogenesis, once edema is established, the clearance of pulmonary fluid is effected by active epithelial Na+ transport out of the alveoli (18). Vectorial movement of Na+ in alveolar epithelial cells depends on the concerted activity of ion transport proteins localized within structurally and functionally distinct plasma membrane domains: Na+-K+-ATPase, confined to the basolateral side, and various ion transporters located at the apical domain of the cell (34). The electrochemical gradient generated by the activity of Na+-K+-ATPase is responsible for the vectorial transport of Na+ from the air space, with water following isosmotically (39, 40, 42, 43).

The alveolar epithelium comprises two distinct cell populations: type I cells, which are thin and elongated and have not been studied in detail due to technical difficulties in isolation, and type II cells, which have been extensively studied. Alveolar epithelial type II (ATII) cells are cuboidal, and their major distinguishing characteristic is their capacity for synthesis and secretion of pulmonary surfactant. In addition to serving as surfactant storage sites, when ATII cells are isolated and cultured on plastic in serum-containing medium, they express the Na+-K+-ATPase alpha 1- and beta 1-subunit isoforms for up to 4 days in culture and maintain active Na+ transport (28, 33). Monolayers of ATII cells generate short-circuit currents and Na+ fluxes that are ouabain inhibitable (23). Increased edema clearance is associated with higher Na+-K+-ATPase activity (31) in alveolar epithelial cells, further supporting the role of this cell population in Na+ and water transport accross the alveolar epithelium.

Catecholamines increase lung fluid clearance via beta -adrenergic-mediated stimulation of active Na+ transport across lung epithelial cells (1). The cellular mechanisms involved are not yet fully elucidated, although they probably include changes in apical Na+ channels (29, 44). In addition, it has been recently proposed that alveolar cell Na+-K+-ATPase plays an important role in lung edema clearance by enhancing active Na+ transport (31). Several intracellular signaling messengers such as cAMP and protein kinase C, which modulate Na+-K+-ATPase activity in other epithelia (5, 6), have been shown to affect fluid transport across alveolar epithelial cells (36, 41).

The purpose of this study was to determine whether Na+-K+-ATPase in ATII cells undergoes acute changes in its catalytic activity in response to short-term beta -adrenergic-receptor stimulation by isoproterenol (Iso) and, if so, to explore possible mechanisms involved in this effect.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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Materials. Phallacidin, Iso, forskolin (FSK), brefeldin A, Tris-ATP, and the 20-residue cAMP-dependent protein kinase inhibitor (IP20) were obtained from Sigma (St. Louis, MO). [gamma -32P]ATP and [3H]ouabain were from Amersham (Arlington Heights, IL). [32P]orthophosphate was purchased from NEN Life Science Products. Rhodamine-phalloidin was from Molecular Probes (Eugene, OR). Elastase was from Worthington Biochemical (Freehold, NJ). The Na+-K+-ATPase alpha -antibody was a generous gift from Dr. M. Caplan (Yale University, New Haven, CT), and the Rab 5 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation and culture of alveolar epithelial cells. ATII cells were isolated from specific pathogen-free male Sprague-Dawley rats (200-225 g) as previously described (15, 28, 33). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (30 U/ml). ATII cells were purified by differential adherence to IgG-pretreated dishes, and cell viability was assessed by trypan blue exclusion (>95%). Cells were suspended in Dulbecco's modified Eagle's medium (DMEM; Irvine Scientific, Santa Ana, CA) containing 10% fetal bovine serum with 2 mM L-glutamine, 40 µg/ml of gentamicin, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. For studies of [gamma -32P]ATP hydrolysis in intact alveolar epithelial cells and preparation of membranes for Western blot analysis, 10 ml of the cell suspension (106 cells/ml) were added to 100-mm dishes (Becton Dickinson). For studies evaluating the cytoskeleton structure, ATII cells were plated onto glass coverslips at 2 × 105 cells/coverslip. Cells were incubated in a humidified atmosphere of 5% CO2-95% air at 37°C. Identification of ATII cells was based on the presence of lamellar inclusions. Lamellar bodies were stained with Papanicolaou stain (22), tannic acid (27), and alkaline phosphatase (17).

Determination of Na+-K+-ATPase activity in intact cells. Na+-K+-ATPase activity was determined as described before (4). Briefly, after the cell suspensions were preincubated with the desired agonists at room temperature, they were placed on ice, and aliquots (~10 µg of protein) were transferred to the Na+-K+-ATPase assay medium (final volume 100 µl) containing (in mM) 50 NaCl, 5 KCl, 10 MgCl2, 1 EGTA, 50 Tris · HCl, and 7 Na2ATP and [gamma -32P]ATP (specific activity 3,000 Ci/mmol) in tracer amounts (3.3 nCi/µl). Cells were transiently exposed to a thermic shock (10 min at -20°C) to render the membranes permeable to ATP. The samples were then incubated at 37°C for 15 min, and the reaction was terminated by the addition of 700 µl of a TCA-charcoal (5:10% wt/vol) suspension and rapid cooling to 4°C. After the charcoal phase containing the unhydrolyzed nucleotide was separated (12,000 g for 5 min), the liberated 32P was counted in an aliquot (200 µl) from the supernatant. Na+-K+-ATPase activity was calculated as the difference between the test samples (total ATPase activity) and the samples assayed in the same medium but devoid of Na+ and K+ and in the presence of 4 mM ouabain (ouabain-insensitive ATPase activity).

Determination of ouabain binding. Ouabain binding was performed in isolated membranes prepared from ATII cells incubated under different protocols in situ or in culture dishes, thus maintaining the polarity of the cells. Ouabain binds to Na+-K+-ATPase in its phosphorylated configuration. In the present study, we used vanadate, an inhibitor with a higher affinity for the phosphorylation site than ATP or phosphate (16), to block the pump in this configuration and to determine the [3H]ouabain bound. To evaluate total and nonspecific [3H]ouabain binding, cultured ATII cells were randomly divided into two groups. In addition, three blanks (incubated without cells) were used in each assay for background determination. Results were calculated and are expressed as picomoles of bound [3H]ouabain per milligram of protein as previously reported (16, 19, 33). Total [3H]ouabain binding was determined in a medium containing (in mM) 250 sucrose, 3 MgSO4, 3 Na2HPO4, 1 NaVO3, and 10 Tris · HCl and 10-4 M [3H]ouabain (25 Ci/mmol), pH 7.2. Nonspecific binding was determined with an identical incubation medium containing a 100-fold excess of unlabeled ouabain. The binding reaction was allowed to proceed for 30 min at 37°C. Unbound [3H]ouabain was removed by rapidly washing the cells three times with ice-cold sample buffer.

Isolation of endosomes. Early and late endosomes were fractionated on a flotation gradient with essentially the technique described by Gorvel et al. (20). ATII cells in suspension (1.5 mg protein/ml) were incubated with either 1 µM Iso, 10 µM FSK, or vehicle at room temperature for 15 min. The incubation was terminated by transferring the samples to ice and adding cold homogenization buffer containing 250 mM sucrose and 3 mM imidazole, pH 7.4. The cells were gently homogenized with a motor pestle homogenizer (15-20 strokes) to minimize damage of the endosomes, and the samples were subjected to a brief (5-min) centrifugation (4°C at 3,000 g). The postnuclear supernatant was adjusted to 40.6% sucrose and loaded (1.5 ml) at the bottom of a 5.0-ml centrifuge tube to which 16% sucrose (1.5 ml) in 3 mM imidazole and 0.5 mM EDTA in deuterium oxide, 10% sucrose in the same buffer (1 ml), and, finally, homogenization buffer (1 ml) were added sequentially. The samples were centrifuged (1 h at 110,000 g) in a Beckman SW 50.1 rotor. Early endosomes were collected at the homogenization buffer-10% sucrose interface and the late endosomes at the 10% sucrose-16% sucrose interface.

Preparation of basolateral plasma membranes. After incubation with the desired agonists, the ATII cells were homogenized, and the basolateral plasma membranes (BLMs) were prepared as described before (12, 21).

Phosphorylation and immunoprecipitation of the Na+-K+-ATPase alpha -subunit in intact ATII cells. Chibalin et al. (12) recently quantitated phosphorylation of the alpha -subunit in a renal cell line (OK), and we have utilized identical procedures in these experiments. Briefly, ATII cells (2.0-2.5 mg protein/dish) were labeled during 2.5 h at 37°C in a buffer containing (in mM) 120 NaCl, 5 KCl, 4 NaHCO3, 1 CaCl2, 1 MgSO4, 0.2 NaH2PO4, 0.15 Na2HPO4, 5 glucose, 10 lactate, 1 pyruvate, and 20 HEPES, 1% bovine serum albumin, pH 7.45, and 100 µCi/ml of [32P]orthophosphate. The cells were labeled and incubated with different agonists in culture dishes to preserve the polarized distribution of different ion transport proteins. The incubation with Iso was performed at room temperature according to protocols similar to those described for Na+-K+-ATPase activity. The incubation was terminated by removing the medium, adding immunoprecipitation buffer [100 mM NaCl, 50 mM Tris · HCl, 2 mM EGTA, 10 mM NaF, 30 mM Na4O7P2, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml of leupeptin, 4 µg/ml of aprotonin, and 1% Triton X-100, pH 7.45], and placing the samples on ice. The cells were disrupted by gentle homogenization. An aliquot was removed for SDS-PAGE analysis of the total pattern of phosphorylation.

Immunoprecipitation of the Na+-K+-ATPase alpha -subunit was performed as described by Carranza et al. (9). Briefly, aliquots (200 µg of protein) were incubated overnight at 4°C with 50 µl of rabbit polyclonal antibody and the simultaneous addition of excess protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden). Samples were washed (four times) with 1 ml of cold immunoprecipitation buffer, resuspended in 100 µl of sample buffer, and analyzed by SDS-PAGE with the Laemmli buffer system (24). Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA), and autoradiography was performed with Kodak film (Rochester, NY).

F-actin staining. ATII cells were lightly fixed in a 3.7% formaldehyde buffer for 15 min at 22°C and thereafter washed with PBS. The cells were incubated for 3 min with permeabilization buffer containing 1% Triton X-100, 0.1 mM PMSF, 0.6 M KCl, and 10 mM PBS. After permeabilization, the cells were washed with PBS and exposed to rhodamine-phalloidin for 30 min at 22°C, and after being washed with PBS, they were mounted on slides for visualization with a fluorescent microscope.

Miscellaneous. Cellular cAMP levels were determined with a commercial kit (cAMP enzyme immunoassay, Amersham) used as recommended by the manufacturer. Protein separation was performed by SDS-PAGE and Western blot analysis as previously described (3, 24). Protein determination was performed according to Bradford (8) with a conventional dye reagent (Bio-Rad, Richmond, CA).

Statistical analysis. Comparisons were performed with the paired Student's t-test. ANOVA with Tukey's test was used to analyze the cAMP data. P values < 0.05 were considered significant.

    RESULTS
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Procedures
Results
Discussion
References

ATII cells after 2 days in culture were resuspended and incubated at room temperature with 1 µM Iso for 15 min in the presence and absence of IP20 (Fig. 1A). Na+-K+-ATPase activity in nonstimulated ATII cells was 97 ± 7 nmol Pi · mg protein-1 · min-1 (n = 5). Iso stimulated Na+-K+-ATPase activity, and this effect was abolished in the presence of 10 nM IP20, which by itself did not significantly change Na+-K+-ATPase activity. Consistent with these results, incubation of ATII cells with Iso was associated with a dose-dependent increase in the cellular levels of cAMP (Fig. 1B).


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Fig. 1.   Na+-K+-ATPase activity (A) and cellular cAMP levels (B) in alveolar type II (ATII) cells incubated with isoproterenol (Iso). A: cells were incubated at room temperature with 1 µM Iso in presence and absence of 10 nM 20-residue cAMP-dependent protein kinase inhibitor (IP20). C, control (vehicle-incubated cells); prot, protein. Each bar is mean ± SE of 5 determinations performed independently (separate cell isolations) and in duplicate. B: cAMP enzyme immunoassay system that utilizes a peroxidase-labeled cAMP conjugate was used to measure cellular cAMP levels. [Isoproterenol], Iso concentration. Each data point is mean ± SE of 4 independent determinations performed in duplicate. * P < 0.05 compared with control.

To determine whether an increase in cellular cAMP (by direct activation of adenylyl cyclase) affects Na+-K+-ATPase activity, ATII cells were also incubated with 10 µM FSK for 15 min at room temperature. FSK significantly increased Na+-K+-ATPase activity as measured by [gamma -32P]ATP hydrolysis (Fig. 2A) as well as by [3H]ouabain binding (Fig. 2B).


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Fig. 2.   Effect of forskolin (FSK) on Na+-K+-ATPase activity (A) and ouabain binding (B) in ATII cells. ATII cells were incubated in presence and absence (vehicle) of 10 µM FSK. Each bar is mean ± SE of 5 experiments for Na+-K+-ATPase activity and 6 experiments for ouabain binding performed in triplicate. Significant differences compared with vehicle.

Because Na+-K+-ATPase activity is stimulated by cAMP in several tissues (2, 14, 26), including the lung epithelia (41), it has been suggested that this effect could be mediated by direct phosphorylation of the alpha -subunit. We therefore examined in ATII cells metabolically labeled with 32P whether incubation with Iso resulted in increased phosphorylation of the catalytic alpha -subunit. Immunoprecipitation of the alpha -subunit from cells treated with Iso revealed no increase in its state of phosphorylation compared with that from vehicle-treated cells (Fig. 3). Alternatively, the FSK-induced increase in [3H]ouabain binding within 15 min might suggest that in ATII cells cAMP stimulates Na+-K+-ATPase activity by increasing the number of Na+-K+ pump units in the plasma membrane.


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Fig. 3.   Effect of Iso on state of Na+-K+-ATPase alpha -subunit phosphorylation in ATII cells. A: Coomassie blue staining and total phosphorylation in ATII cell lysates (100 µg of protein loaded in each lane). V, vehicle. Nos. at left, molecular mass. B: Western blot and autoradiography of immunoprecipitated alpha -subunit. This experiment is representative of 3 independent cell isolations.

Stabilizing the cortical cytoskeleton with the fungal toxin phallacidin has proven to be an important tool in studying the relevance of actin dynamics in several physiological processes (38). The next series of experiments was therefore performed in ATII cells pretreated with phallacidin (1 µM) overnight. This treatment did not affect the cell morphology or the distribution of actin polymers within the cell cytoplasm (Fig. 4). However, monolayers pretreated with phallacidin did not exhibit the increments in Na+-K+-ATPase activity and [3H]ouabain binding (Fig. 5, A and B, respectively) observed in response to FSK alone, even though the ability of FSK to increase the cellular levels of cAMP was not affected (Fig. 5C).


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Fig. 4.   Effect of phallacidin treatment on ATII cell morphology and F-actin content. Representative photomicrographs of phase-contrast and F-actin fluorescence in ATII cells incubated overnight in presence (+) and absence (-) of 1 µM phallacidin. Bars, 200 µm for phase contrast and 25 µm for F-actin.


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Fig. 5.   Effect of FSK on Na+-K+-ATPase activity (A), ouabain binding (B), and cellular cAMP levels (C) in phallacidin-treated ATII cells. Cells were incubated overnight with 1 µM phallacidin (solid bars); thereafter, they were incubated in presence and absence (vehicle) of 10 µM FSK. Open bars, without phallacidin. Each bar is mean ± SE of 5 (A and B) and 4 (C) independent cell isolations. Determinations were performed in triplicate. * P < 0.01 compared with respective vehicle.

We next evaluated the effect of Iso and FSK on Na+-K+-ATPase alpha -subunit abundance in the BLM using Western blot analysis. BLMs were prepared from monolayers incubated with 1 µM Iso or 10 µM FSK for 15 min at room temperature. Iso and FSK increased the alpha -subunit abundance to a similar extent, and this effect was completely blocked by pretreatment with phallacidin (Fig. 6).


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Fig. 6.   Effect of Iso and FSK on Na+-K+-ATPase alpha -subunit abundance in basolateral plasma membranes from ATII cells. Top: representative Western blot. Equal amounts of protein (5 µg) were loaded in each lane. Bottom: quantitatve densitometric scans of 4 experiments. Values are means ± SE. * P < 0.05 compared with (+)phallacidin.

Further support for traffic of Na+-K+-ATPase molecules as a mechanism for increased activity was provided by studies performed in the presence of the fungal toxin brefeldin A. Preincubation of ATII cells with brefeldin A (5 µg/ml at room temperature for 20 min) inhibited Na+-K+-ATPase activity (vehicle: 61.2 ± 10 nmol Pi · mg protein-1 · min-1, n = 10; brefeldin A: 40.7 ± 5 nmol Pi · mg protein-1 · min-1, n = 5; P < 0.05). Under this condition, Iso (15 min at room temperature) failed to stimulate enzyme activity (Iso + brefeldin A: 39.5 ± 4 nmol Pi · mg protein-1 · min-1; n = 4 experiments). The effects of Iso and brefeldin A on Na+-K+-ATPase alpha -subunit abundance in BLMs were also evaluated in ATII monolayers (Fig. 7). Iso increased Na+-K+-ATPase alpha -subunit abundance, and this effect was blocked by brefeldin A treatment.


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Fig. 7.   Na+-K+-ATPase alpha -subunit distribution in basolateral plasma membranes from ATII cells preincubated with 5 µg/ml of brefeldin A (20 min at 23°C) and incubated with and without 1 µM Iso as shown by a representative (n = 3 membranes) Western blot analysis. Equal amounts of protein (5 µg) were loaded.

The stimulatory effect of Iso on Na+-K+-ATPase alpha -subunit abundance in the BLM appeared to be independent of Na+ permeability because it was not altered by coincubation with 10 µM amiloride (Fig. 8). In addition, the stimulatory effect of Iso (210 ± 14% of control value; P < 0.05; n = 5 experiments) on Na+-K+-ATPase activity was also not abolished by coincubation with amiloride (238 ± 38% of control value; n = 4 experiments), and amiloride alone did not affect Na+-K+-ATPase activity (95.7 ± 25% of control value; n = 4 experiments).


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Fig. 8.   Na+-K+-ATPase alpha -subunit distribution in ATII cells incubated with and without Iso in presence and absence of 1 µM amiloride (Amil), an Na+-channel blocker. Top: representative Western blot analysis. Equal amounts of protein (5 µg) were loaded in each lane. Bottom: quantitative densitometric scans of 4 experiments. Values are means ± SE. * P < 0.05 compared with Amil alone.

The increased alpha -subunit abundance is not likely to represent increased de novo synthesis of Na+-K+-ATPase molecules because the Iso effect occurs within 15 min. Thus we hypothesized that the increased number of Na+-K+-ATPase molecules within the BLM may be caused by recruitment of existing units from intracellular compartments. We therefore prepared early and late endosomes from ATII cells, the purity and identity of which were confirmed by enrichment of Rab 5- and mannose 6-receptor immunoreactivity, respectively (11). Both populations of endosomes contained Na+-K+-ATPase alpha -subunits (Fig. 9). In late but not in early endosomes prepared from Iso-treated ATII cells, there was a significant decrease in alpha -subunit abundance, with a concomitant increase in their presence in BLMs from the same cell population (Fig. 9).


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Fig. 9.   Na+-K+-ATPase alpha -subunit abundance in early (EE) and late (LE) endosomes (E) prepared from Iso-treated ATII cells. Cells were incubated with 1 µM Iso, and EE and LE were prepared as described in EXPERIMENTAL PROCEDURES. Basolateral membranes (BLM) were also prepared from the same cell isolation (n = 2). Top: representative Western blot. Equal amounts of protein (3 µg) were loaded in each lane. Bottom: quantitative densitometric scans of 4 experiments. Values are means ± SE. * P < 0.05 compared with vehicle.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we demonstrate that Iso increases Na+-K+-ATPase activity of ATII cells independently of Na+ permeability and that this effect is mediated by increasing the number of Na+-K+-ATPase molecules within the BLM. The stimulatory action of Iso is associated with increased levels of cAMP but does not involve phosphorylation of the Na+-K+-ATPase alpha -subunit.

Although regulation of ATII cell Na+-K+-ATPase activity by beta -adrenergic agonists has been previously suggested (41), the intrinsic mechanisms of this regulation as well as the intracellular signaling systems involved have not yet been completely elucidated.

Here we report that short-term (15-min) incubation with 1 µM Iso stimulated Na+-K+-ATPase activity in ATII cells (measured as hydrolytic activity in cell suspensions or ouabain binding in cell monolayers) and that this effect was associated with a significant increase in the cellular levels of cAMP (Fig. 1B). Furthermore, the stimulatory effect of Iso was blocked by a cAMP-dependent protein kinase inhibitor. However, Suzuki et al. (41) could not demonstrate a direct correlation between the increase in Na+-K+-ATPase activity and the rise in cellular cAMP levels. In that report, a cAMP analog increased Na+-K+-ATPase activity in alveolar cells cultured for 2 and 5 days, whereas the beta -adrenergic agonist terbutaline, although able to increase cAMP levels at both intervals, failed to stimulate Na+-K+-ATPase activity in cells cultured for 2 days. Our experiments were performed in resuspended ATII cells (hydrolytic activity) or cell monolayers (ouabain binding) after 2 days in culture, and we indeed found a correlation between the changes in Na+-K+-ATPase activity and the increase in cAMP (see Figs. 1 and 2). At present, we do not have an explanation for the differences between these two studies. Furthermore, the effect of Iso was blocked by a cAMP-dependent protein kinase inhibitor.

Short-term regulation of Na+-K+-ATPase activity by hormones and the signaling systems involved have been extensively studied (5, 6). Accumulating evidence indicates that most receptor signals converge in the activation of protein kinases. Phosphorylation of the purified Na+-K+-ATPase alpha -subunit preparations by both protein kinase C and cAMP-dependent protein kinase resulted in a significant change in its catalytic activity (3, 13, 25). In addition, studies in intact cells also demostrated that regulation of Na+-K+-ATPase activity may be accomplished by phosphorylation of its catalytic subunit. Specifically, activation of cAMP kinase in intact cells has been associated with increased Na+-K+-ATPase activity (10). In our studies, Iso stimulation of Na+-K+-ATPase activity, although associated with increased cellular levels of cAMP and presumably activation of a cAMP-dependent protein kinase, was not due to increased phosphorylation of the Na+-K+-ATPase catalytic subunit (Fig. 3). The lack of alpha -subunit phosphorylation suggests that cAMP and cAMP-dependent kinase may be necessary at other stages of Na+-K+ pump regulation, possibly during the unit's traffic to the BLM.

An alternative mechanism by which Iso could stimulate Na+-K+-ATPase activity is by increasing the number of functioning units in the plasma membrane. This effect could be brought about by alterations in the rate of protein synthesis, consequently leading to changes in enzymatic activity. However, this process (which usually occurs over a period of hours) cannot explain the observed Iso effect, which occurred within 15 min. Because stimulation of Na+-K+-ATPase activity was associated with an increase in the number of units at the cell surface (Fig. 2B, ouabain binding, and Fig. 6, alpha -subunit abundance), we reasoned that pump units present in one or more intracellular compartments may have been recruited for insertion in the plasma membrane. Contrary to Suzuki et al. (41), we did find an increase in alpha -subunit abundance at the BLM. Although speculative, a possible explanation why Suzuki et al. did not observe such an increase is because the high-speed centrifugation utilized in their protocols to obtain the membrane preparations could have caused the inclusion of endosomes and other organelles in the isolated material, thus making it difficult to distinguish between the proportion of molecules that are incorporated in the basolateral membranes and those from the intracellular compartments.

We have demonstrated in renal epithelial cells the presence of defined intracellular compartments in which Na+-K+-ATPase is located (11). On incubation with dopamine, the resultant decrease in Na+-K+-ATPase activity was associated with a stepwise transfer of Na+-K+-ATPase alpha - and beta -subunits from the plasma membrane into early and late endosomes via a clathrin vesicle-dependent mechanism (11). In the present study, we have also identified Na+-K+-ATPase subunits within these compartments in ATII cells, although the transfer seems to occur in the opposite direction, i.e., from late endosomes to the plasma membrane. In cells incubated with Iso for 15 min, there is an increased incorporation of Na+-K+-ATPase alpha -subunits in the plasma membranes, associated with a decrease in their abundance in the late endosomal compartment without a significant change in early endosomes (see Figs. 6 and 9). It is possible that the alpha -subunits present in the early endosomes are there as the consequence of a constitutive or a regulated endocytic pathway as demonstrated in renal cells (11), whereas in late endosomes, they may represent a pool that can recycle to the plasma membrane on rapid demand. This pool could be replenished by newly synthesized Na+-K+-ATPase subunits or by those from the endocytic pathway that have not proceeded to degradation in lysosomes.

The reason(s) why the percent increase of the alpha -subunit in the BLM (~80%) does not correspond to its decrease in the late endosomes (~30%) is not entirely clear. An important factor determining the relative changes in Na+-K+-ATPase in the two compartments is the relative size of these compartments accessible to analysis. A likely explanation, although speculative at this point, could therefore be that different recoveries are obtained during the isolation of endosomes and BLMs (because, after the flotation gradient fractionation used to separate endosomes, the fraction containing BLMs was further enriched with Percoll gradient centrifugation), making these determinations (percent changes) semiquantitative. However, when fractions were prepared by the same method (in kidney cells), we found a decrease in alpha -subunit abundance in the fraction containing BLMs comparable to the increase in the fraction containing endosomes (11). Another possibility to be considered is that of "serial compartmentalization" of alpha -subunit traffic between the late endosomes and the plasma membrane: alpha -subunits will not reach the plasma membrane solely from the late endosomes but also from intermediate compartments yet unidentified or not accessible to isolation. Also, from our studies, it cannot be excluded that there may be additional mechanisms responsible for the effect under discussion.

Additional evidence suggesting that Iso stimulation of Na+-K+-ATPase activity is accomplished by favoring the movement of pump molecules from intracellular pools to the plasma membrane is provided by the finding that this process was inhibited by stabilizing the actin cytoskleton with phallacidin. The mechanisms underlying the regulatory role of actin cytoskeleton dynamics in these transport events are still unclear, but one possibility would be by permitting the motion of the late endosomes containing Na+-K+-ATPase toward the BLM and their fusion with it. Also, incubation with the fungal toxin brefeldin A, which prevents anterograde transport, abolished the increased alpha -subunit abundance in BLMs induced by Iso. Therefore, this study also raises the possibility that Iso might regulate the actin cytoskeleton to favor the traffic of Na+-K+ pump molecules to their cellular destination.

Vectorial transport of Na+ in the alveolar epithelium is critical for modulation of alveolar fluid clearance (31, 32, 37). Administration of beta -adrenergic agonists has been shown to increase active Na+ transport and lung edema clearance (35), but the mechanisms whereby beta -adrenergic stimulation results in increased lung edema clearance are only partially understood. It is postulated that activation of apical Na+ channels (29, 44) and stimulation of Na+-K+-ATPase activity are the cellular mechanisms involved (42). This coordinated transport would entail changes in vectorial movement of Na+ without affecting its intracellular concentration as reported for other tissues (7), thus preventing further changes in cellular homeostasis.

The concept that Iso would independently affect Na+ permeability and active transport is supported by the present results, which suggest that the action of Iso on Na+-K+-ATPase activity is not mediated by, or dependent on, an increase in intracellular Na+. Amiloride, at concentrations that should have blocked the majority of Na+ entry pathways, did not prevent Iso-dependent stimulation of Na+-K+-ATPase activity or the increase in alpha -subunit abundance in the BLM (Fig. 8). This observation and that of a previous report on ATII cells (41) differ from the situation in renal proximal tubule cells where Iso-dependent regulation of Na+-K+-ATPase activity appears to be the result of increased Na+ permeability (39).

In conclusion, this study demonstrates that beta -adrenergic agonists (Iso) regulate Na+-K+-ATPase activity in transporting epithelia (such as ATII cells) by increasing the number of Na+-K+-ATPase units in the BLM. This regulation is rapid and probably does not involve synthesis of new molecules. Rather, the new Na+-K+-ATPase units incorporated in the BLM are apparently recruited from defined intracellular compartments, e.g., the late endosomes. Once in the BLM, these newly inserted transport proteins contribute to the overall increase in cellular Na+-K+-ATPase activity and consequently to vectorial Na+ flux across the alveolar epithelium.

    ACKNOWLEDGEMENTS

We thank Chris Waters for help with this work.

    FOOTNOTES

This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-48129 (to J. I. Sznajder); a National Research Science Award from the National Institutes of Health (to K. M. Ridge); an American Heart Association grant (to J. I. Sznajder); and a Swedish Medical Research Council grant (to A. M. Bertorello)

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. §1734 solely to indicate this fact.

Address for reprint requests: A. M. Bertorello, Dept. of Molecular Medicine L6B:02, Karolinska Hospital, S-171 76 Stockholm, Sweden.

Received 22 April 1998; accepted in final form 2 September 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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