Analytical isolation of plasma membranes of intestinal epithelial cells: Identification of Na,K-ATPase rich membranes and the distribution of enzyme activities

Summary A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and cole material. Sucrase specific activity in the purified brush border plasma membrane was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochromec reductase, nonspecific esterase, -glucoronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity.Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent³⁵S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein.Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Analytical isolation of plasma membranes of intestinal epithelial cells: identification of Na, K-ATPase rich membranes and the distribution of enzyme activities.

A procedure was developed for the analytical isolation of brush border and basal lateral plasma membranes of intestinal epithelial cells. Brush border fragments were collected by low speed centrifugation, disrupted in hypertonic sorbitol, and subjected to density gradient centrifugation for separation of plasma membranes from nuclei and core material. Sucrase specific activity in the purified brush border plasma membranes was increased fortyfold with respect to the initial homogenate. Basal lateral membrane were harvested from the low speed supernatant and resolved from other subcellular components by equilibrium density gradient centrifugation. Recovery of Na, K-ATPase activity was 94%, and 61% of the recovered activity was present in a single symmetrical peak. The specific activity of Na, K-ATPase was increased twelvefold, and it was purified with respect to sucrase, succinic dehydrogenase, NADPH-cytochrome c reductase, nonspecific esterase, beta-glucuronidase, DNA, and RNA. The observed purification factors are comparable to results reported for other purification procedures, and the yield of Na, K-ATPase is greater by a factor of two than those reported for other procedures which produce no net increase in the Na, K-ATPase activity. Na, K-ATPase rich membranes are shown to originate from the basal lateral plasma membranes by the patterns of labeling that were produced when either isolated cells or everted gut sacs were incubated with the slowly permeating reagent 35S-p-(diazonium)-benzenesulfonic acid. In the former case subsequently purified Na, K-ATPase rich and sucrase rich membranes are labeled to the same extent, while in the latter there is a tenfold excess of label in the sucrase rich membranes. The plasma membrane fractions were in both cases more heavily labeled than intracellular protein. Alkaline phosphatase and calcium-stimulated ATPase were present at comparable levels on the two aspects of the epithelial cell plasma membrane, and 25% of the acid phosphatase activity was present on the basal lateral membrane, while it was absent from the brush border membrane. Less than 6% of the total Na, K-ATPase was present in brush border membranes.     

Stimulation-associated redistribution of Na,K-ATPase in rat lacrimal gland

Summary To test the possibility that stimulation of secretion leads Na,K-ATPase to be recruited from cytoplasmic pools and inserted into basal-lateral plasma membranes, we surveyed the subcellular distributions of Na, K-ATPase in resting and stimulated fragments of rat exorbital lacrimal gland. After a two-dimensional separation procedure based on differential sedimentation and density gradient centrifugation, we defined sixdensity windows, which differ from one another in their contents of biochemical markers. The membranes equilibrating inwindow I could be identified as a sample of basal-lateral membranes; in resting preparations these membranes contained Na,K-ATPase enriched 16.6-fold with respect to the initial homogenates.Windows II throughVI contained various cytoplasmic membrane populations; these accounted for roughly 80% of the total recovered Na,K-ATPase activity. Thirty-minute stimulation with 10 m carbachol caused a 1.4-fold increase (P<0.05) in the total Na,K-ATPase content ofwindow I; this increase could be largely accounted for by a 1.7-fold decrease in the total Na,K-ATPase content ofdensity window V. Acid phosphatase activity also redistributed following stimulation, but it was recruited from a different source, and it was inserted into membranes equilibrating inwindows II andIII as well as into the membranes ofwindow I.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Complex subcellular distributions of enzymatic markers in intestinal epithelial cells

Summary Current procedures for isolating intestinal epithelial cell surface and intracellular membranes are based on the assumption that each organelle is marked by some unique constitutent. This assumption seemed inconsistent with the dynamic picture of subcellular organization emerging from studies of membrane turnover and recycling. Therefore, we have designed an alternative fractionation which is independent ofa priori marker assignments. We subjected mucosal homogenates to a sequence of separations based on sedimentation coefficient, equilibrium density, and partitioning in aqueous polymer twophase systems. The resulting distributions of protein and enzymatic markers define a total of 17 physically and biochemically distinct membrane populations. Among these are: basal-lateral membranes, with Na,K-ATPase enriched 21-fold; brush-border membranes, with alkaline phosphatase enriched as much as 38-fold; two populations apparently derived from the endoplasmic reticulum; a series of five populations believed to have been derived from the Golgi complex; and a series of five acid phosphatase-rich populations which we cannot identify unequivocally. Each of the five enzymatic markers we have followed is associated with a multiplicity of membrane populations. Basallateral, endoplasmic reticulum, and Golgi membranes contain alkaline phosphatase at the same specific activity as the initial homogenate. Similarly, Na,K-ATPase appears to be associated branes at specific activities two-to seven-fold that of the initial homogenate.     

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

Summary The effect of phospholipase A₂ and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A₂ (PLA₂) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (P_i+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA₂, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and P_i+Mg media. Other detergents were ineffective.The ability of PLA₂ to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the P_i+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as P_i-oriented ouabain sites are inhibited by PLA₂.     

Subcellular distribution of tissue kallikrein and Na,K-ATPase α-subunit in rat parotid striated duct cells

Intracellular protein distribution and sorting were examined in rat parotid striated duct cells, in which tissue kallikrein is apical, and Na,K-ATPase is basolateral. Electron-microscopic immunogold cytochemistry, with both polyclonal and monoclonal antibodies, demonstrated these enzymes at opposite poles of the cells and in distinct intracellular sites. Kallikrein was found within apical secretory granules, whereas Na,K-ATPase was present on basolateral cell membranes. In addition, kallikrein was localized throughout cisternae of all Golgi profiles, whereas Na,K-ATPase (-subunit) was found only in small peripheral vesicles and/or lateral cisternal extensions of a basal subset of Golgi profiles. These differences in the subcellular distribution of the two marker antigens were most clearly seen with double immunogold labelling. Our results suggest that kallikrein, an apical, regulated secretory protein, and Na,K-ATPase, a basolateral, constitutively transported membrane protein, are segregated at (or prior to) the level of the Golgi apparatus rather than in the trans-Golgi network (TGN), as was expected.Abbreviations ATP adenosine tri-phosphate - HBSS Hanks' balanced salt solution - GaM goat anti-mouse - GaR goat anti-rabbit - PBS phosphate-buffered saline - RaM rabbit anti-mouse - RER rough endoplasmic reticulum - TGN trans-Golgi network     

Isolation of plasma membranes from bovine corpus luteum possessing adenylate cyclase, 125I-hCG binding and NaK-ATPase activities

Plasma membranes from bovine corpora lutea have been purified by sucrose density gradient centrifugation. The purified membranes, in addition to binding ¹²⁵I-hCG, also possess hCG-stimulated adenylate cyclase and NaK-ATPase. The relative purification of ¹²⁵I-hCG binding, adenylate cyclase and NaK-ATPase on the basis of the specific activities in the whole homogenate were 7.8, 6.4 and 2.6, respectively. The presence of both the hormone sensitive adenylate cyclase and ¹²⁵I-hCG binding activities suggest that these plasma membranes might possess the ‘receptor’ for gonadotropin.     

Lateral mobility of Na,K-ATPase and membrane lipids in renal cells. Importance of cytoskeletal integrity

Because membrane fluidity is an important determinant of membrane function, the lateral diffusion rate (D _L ) of the membrane protein Na,K-ATPase was determined in intact renal proximal tubule epithelial cells by the technique of fluorescence redistribution after photobleaching (FRAP). In normal cells the D _L of Na,K-ATPase in the basal membrane was 3.31×10^–10 cm²/ sec. Treatment with cytochalasin D to promote actin filament depolymerization caused a sevenfold increase in D _L . Exposure of cells to a Ca²⁺-free medium or to hypoxia and reoxygenation, which have similar disruptive effects on the cytoskeleton, also caused increases in D _L . Disruption of actin microfilament structure also increased the mobile fraction of Na,K-ATPase. Using a confocal laser microscopic technique only 14.9% of total Na,K-ATPase was observed to reside in the apical membrane domain of normal cells. Microfilament depolymerization caused this fraction to increase to 47.7%. Thus, the translocation of Na,K-ATPase from the basolateral to the apical domain induced by cytoskeletal protein dysfunction was enabled by an increased rate of lateral diffusion of Na,K-ATPase. The behavior of a variety of membrane lipids following actin depolymerization was more heterogeneous. Some lipids showed a similar increase in D _L whereas others showed very little dependence upon the cytoskeleton for lateral restraint.This work was supported by an American Heart Association Grant-in-Aid, an extramural grant from Baxter Healthcare Corporation, and NIH Shared Instrument Grant RR-05877. We thank Dr. J. Carlos Manivel for performing the electron microscopic studies.This paper was prepared with the technical assistance of Xing-Xing Luo and Marshalleen Patten.     

Modulation of Na,K-ATPase by Associated Small Transmembrane Regulatory Proteins and by Lipids

The effects of phospholipid acyl chain length (n_c) and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal hydrophobic thickness of the lipid bilayer decreases from n_c = 22 to 18 in the presence of 40 mol% cholesterol. Hydrophobic matching as well as specific interactions of cholesterol with the phosphorylation/dephosphorylation reactions is found to be important. A novel regulatory protein has been identified in Na,K-ATPase membrane preparations from the shark (phospholemmanlike protein from shark, PLMS) with significant homology to phospholemman (PLM), the major protein kinase substrate in myocardium. Both are members of the FXYD gene family. Another member of this family is the Na,K-ATPase subunit indicating that these proteins may be specific regulators of the Na,K-ATPase. A regulatory mechanism is described in which association/dissociation of PLMS with the Na,K-ATPase is governed by its phosphorylation by protein kinases.     

Oxidative Stress (Glutathionylation) and Na,K-ATPase Activity in Rat Skeletal Muscle

Background

Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation) on the Na,K-ATPase in rat skeletal muscle membranes.

Results

Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM) increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05) in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0–10 mM) increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.

Conclusion

This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.

Perspective

Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

Ultrastructural,cyto- and biochemical observations during turnover of plasma membrane in duck salt gland

Summary The mechanism of plasma membrane turnover was investigated using the duckling salt gland as a model system. Feeding fresh water to saltstressed ducklings results in a decrease in the Na, K-ATPase in salt gland to nonstressed levels in about 7 days, as measured by ATP hydrolysis and ³H-ouabain binding. Electron micrographs reveal that this is accompanied by a decrease in plasma membrane infoldings on the basal and lateral borders of gland secretory cells. Simultaneously there is an increase in filamentous material and a rise in acid phosphatase and peptidase activities in these cells. Cytochemistry shows that the acid phosphatase activity is mostly associated with the basal or basolateral regions of secretory cells. These observations could indicate that the removal of plasma membrane components is accomplished by internalization and digestion within the secretory cells.     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Major organ-specific glycoproteins in isolated brain and kidney membranes identified as Na,K-ATPase subunits by combined glycan-, lectin-, and immunoblotting

In the present work combined glycan-, lectin-, and immunoblotting of isolated brain and kidney membranes shows that the and subunits of Na,K-ATPase are the most abundant glycoproteins. Further,Datura stramonium andGalanthus nivalis agglutinins recognize the Na,K-ATPase subunits in a mutually exclusive manner in membranes from human, rabbit and rat brain or human, rabbit, rat, pig and dog kidney indicating the presence of species-independent organ-typical glycoforms. The glycosylation status is not related to the ouabain-sensitivity. Taken together, the data reveals organ-specific glycoforms of Na,K-ATPase which might have roles for organ identification and recognition.Abbreviations NKA Na,K-ATPase (EC 3.6.1.37) - PAGE polyacrylamide gel electrophoresis in dodecylsulfate - Con-A Concanavalin A - DSA Datura stramonium agglutinin - GNA Galanthus nivalis agglutinin - MAA Maackia amurensis agglutinin - PNA Peanut agglutinin - SNA Sambucus nigra agglutinin - WGA Wheat germ agglutinin Abbreviations used in figures K kidney - B brain - Cr Crude - De Detergent-treated - Fe fetuin - Ct creatinase - I-blot immuno-blot - L-blot lectin-blot     

Studies concerning the possible reconstitution of an active cation pump across an artificial membrane

Summary The cortical tissue of rat brain was fractionated through zonal centrifugation in a continuous sucrose density gradient, yielding a variety of morphologically distinct membrane fragments derived from nerve-end particles possessing variable levels of activity of Na, K-dependent Mg-sensitive ATPase (Na, K-ATPase) and other enzymes. Upon addition of certain of the zonal fractions, particularly those rich in the ATPase and acetylcholinesterase activities, to one side of planar artificial membranes, formed from mixtures of oxidized cholesterol and alkanes and bathed in a solution containing sodium, potassium, and magnesium ions, direct current membrane resistance fell from one to three orders of magnitude. Subsequent addition of ATP to the same side of the membrane to which the ATPase was added (thecis side) led to the development of net short-circuit current flow and open-circuit potential across the membrane (thecis side being negative with respect to thetrans side). Development of the short-circuit current and open-circuit potential is dependent upon the presence of all the substrates of Na, K-ATPase as well as that of the enzyme itself. The net current flow is inhibited and the open-circuit potential discharged by the addition of ouabain to thetrans side of the membrane, of phospholipase A to thecis side, or of trypsin to either side of the membrane. These observations provide circumstantial evidence for the reconstitution of the active cation pump across the artificial bilayer. Efforts to effect a similar reconstitution across membranes of this and other compositions employing Na, K-ATPase preparations from beef heart, beef brain, cat brain, human red cells, rabbit kidney, and rat brain microsomes failed.Career Development Awardee of the National Institutes of Health, Grant No. GM 10248.     

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.